Nucleic acid coding for an α-acetolactate synthase from lactococcus and its applications

ABSTRACT

The nucleic acid coding for an α-acetolactate synthase from Lactococcus is provided, as well as vectors containing this nucleic acid and the use of these vectors for transforming microorganisms in which the production of α-acetolactate will be promoted, The nucleic acid comprises one or the other or both of a first segment corresponding to the ilvB gene (which encodes one subunit of α-acetolactate synthase of Lactococcus lactis subsp. lactis) and a second segment corresponding to the ilvN gene (which encodes a second subunit of α-acetolactate synthase of Lactococcus lactis subsp. lactis).

This application is the U.S. national stage application of PCT International Application No. PCT/GB93/02012, filed 27 Sep. 1993, which claims priority of French Patent Application 92/11470, filed 25 September 1992.

The invention relates to a nucleic acid coding for an α-acetolactate synthase, as well as to vectors containing this nucleic acid and to the use of these vectors for transforming microorganisms in which the production of α-acetolactate and diacetyl will be promoted.

Diacetyl is a flavouring sought in the production of food products such as butter, creme fraiche and some cheeses. Conversely, this flavouring is undesirable in the production of other food products such as beer.

A limited number of bacteria, such as Lactococcus lactis subsp. lactis biovar diacetylactis as well as bacteria of the genera Leuconostoc, Pediococcus, Lactobacillus, naturally produce diacetyl; this production takes place from a substrate present in milk in small amounts, citrate; these bacterial strains are capable of metabolizing citrate to pyruvate, which is then converted to acetoin and to diacetyl.

α-Acetolactate is also an intermediate of the pathway of synthesis of the branched amino acids, which are leucine, isoleucine and valine. This pathway has been very widely studied in bacteria, fungi and plants (1, 2, 3, 4). However, the sequences of the assembly of genes involved in this pathway in one and the same microorganism have never been reported.

The regulation of the expression of the genes involved in the pathway of synthesis of the branched amino acids is complex on account of the steps common to the synthesis of these three amino acids, and this pathway is often presented as a model for the study of organization and regulation.

The organization of these genes has been characterized in Escherichia coli (3), Salmonella typhimurium (3), Bacillus subtilis (5, 6, 7), Corynebacterium typhimurium (8) and Staphylococcus aureus (9).

On the E. coli chromosome, the genes are localized in three groups (10). The largest, located at 85 minutes, is organized in one large transcription unit and two small transcription units, comprising the ilvGMEDA, ilvY and ilvC genes, respectively (11, 12); another group, located at 2 minutes, is composed of two transcription units comprising the ilvH and leuACBD genes (13, 14), and the last group, at 82 min, groups together the ilvBN genes in a single transcription unit.

A similar organization is found in other Entero-bacteriaceae.

In B. subtilis, the ilvBN and leuACBD genes are encountered in one region of the chromosome (5) and the ilvAD genes in another region.

Three cloned nonadjacent chromosomal fragments of C. glutamicum carry five genes of the pathway of synthesis of the branched amino acids, ilvCBN, ilvA and ilvE (9).

In S. aureus, genetic mapping shows that eight genes are grouped together in the following order: ilvAB-CD, leuABCD.

The synthesis of the branched amino acids leucine, isoleucine and valine in L. lactis subsp. lactis is a complex pathway involving eight enzymes, four of which are common to the synthesis of the above three amino acids. The synthesis of valine involves only these four, whereas the synthesis of isoleucine and of leucine involves, respectively, one and four additional specific enzymes. In addition, this system is complicated by the fact that the excess of one of the three amino acids interferes in the synthesis of the other amino acids.

The present invention relates to the cloning, characterization and sequencing of the genes involved in the pathway of synthesis of the branched amino acids in L. lactis subsp. lactis. This work was carried out using the strain NCDO2118 (AFRC, Institute of Food Research Reading Laboratory, Shinfield, Reading, Berks, United Kingdom). The genes were characterized by cloning, complementation in E. coli and B. subtilis and sequence analysis. Nine structural genes are assembled on a 12-kb DNA fragment in the following order: leuABCDilvDBNCA.

Among these genes, the invention relates to nucleic acid sequence coding for two polypeptides forming the subunits of a protein possessing α-acetolactate synthase activity; this enzyme is of very special interest since it is responsible, in L. lactis subsp. lactis, for the direct conversion of pyruvate to α-acetolactate independently of the presence of citrate in the medium.

The techniques used for cloning and sequencing the nucleic acid of the invention will be described more particularly in the detailed description of the invention.

Reference will be made in what follows to the tables and figures, wherein:

Table I shows the bacterial strains, plasmids and phages used for cloning the L. lactis subsp. lactis genes involved in the pathway of synthesis of the branched amino acids;

Table II shows a comparison of proteins involved in the pathway of synthesis of the branched amino acids between L. lactis subsp. lactis and different microorganisms;

FIG. 1 shows the structure of the region of L. lactis subsp. lactis DNA carrying the genes involved in the pathway of synthesis of the branched amino acids;

FIGS. 2A-2F show the nucleotide sequence, together with the polypeptide sequence deduced from this nucleotide sequence, of the region of L. lactis subsp. lactis strain NCDO2118 DNA carrying the genes involved in the pathway of synthesis of the branched amino acids; nucleotides 8018 to 9742 of SEQ ID NO: 11 represent the ilvB gene (SEQ ID NO: 12) and encode 575 amino acids as shown (SEQ ID NO: 7); nucleotides 9738 to 10211 of SEQ ID NO: 11 represent the ilvN gene (SEQ ID NO: 13) and encode 158 amino acids as shown (SEQ ID NO: 8);

FIG. 3 shows the alignment of 6 ATP-binding proteins with ORF2 of L. lactis;

FIGS. 4A and 4B show the secondary structures of the major transcript serving as a transcription attenuator.

Research work carried out on the DNA of FIG. 2, and also on the polypeptide deduced from this DNA, leads to the following observations:

Ten genes are present in the region of L. lactis subsp. lactis DNA shown in FIG. 2:

the leuA gene of 1539 nucleotides, located between nucleotides 450 and 1988 of SEQ ID NO: 11 and coding for a polypeptide of 513 amino acids (SEQ ID NO: 1);

the leuB gene of 1041 nucleotides, located between nucleotides 2003 and 3037 of SEQ ID NO: 11 and coding for a polypeptide of 347 amino acids (SEQ ID No: 2 );

the leuC gene of 1380 nucleotides, located between nucleotides 3403 and 4782 of SEQ ID NO: 11 and coding for a polypeptide of 460 amino acids (SEQ ID NO: 3);

the leuD gene of 573 nucleotides, located between nucleotides 4805 and 5377 of SEQ ID NO: 11 and coding for a polypeptide of 191 amino acids (SEQ ID NO: 4);

the orf2 gene of 777 nucleotides, located between nucleotides 5394 and 6170 of SEQ ID NO: 11 and coding for a polypeptide of 259 amino acids (SEQ ID NO: 5);

the ilvD gene of 1710 nucleotides, located between nucleotides 6295 and 8004 of SEQ ID NO: 11 and coding for a polypeptide of 570 amino acids (SEQ ID NO: 6);

the ilvB gene of 1725 nucleotides (SEQ ID NO: 12 which includes the stop codon), located between nucleotides 8018 and 9742 of SEQ ID NO: 11 and coding for a polypeptide of 575 amino acids (SEQ ID NO: 7);

the ilvN gene of 474 nucleotides (SEQ ID NO: 13 which includes the stop codon), located between nucleotides 9738 and 10211 of SEQ ID NO: 11 and coding for a polypeptide of 158 amino acids (SEQ ID NO: 8);

the ilvC gene of 1032 nucleotides, located between nucleotides 10260 and 11291 of SEQ ID NO: 11 and coding for a polypeptide of 344 amino acids (SEQ ID NO: 9);

the ilvA gene of 1323 nucleotides, located between nucleotides 11337 and 12659 of SEQ ID NO: 11 and coding for a polypeptide of 441 amino acids (SEQ ID NO: 10).

The invention relates to any nucleic acid comprising all or part of the DNA sequence bounded by the nucleotides located at positions 8018 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for one or both subunits of a protein capable of converting pyruvate to α-acetolactate; and for any other novel polynucleotide, peptide or protein disclosed herein.

α-Acetolactate synthase of L. lactis subsp. lactis is a protein consisting of two subunits; the first subunit is a polypeptide of 575 amino acids (SEQ ID NO: 7) encoded by the fragment bounded by the nucleotides located at positions 8018 and 9742 of SEQ ID NO: 11 and as shown in FIG. 2, and corresponding to the ilvB gene (SEQ ID NO: 12); the second subunit is a polypeptide of 158 amino acids (SEQ ID NO: 8) encoded by the fragment bounded by the nucleotides located at positions 9738 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2, and corresponding to the ilvN gene (SEQ ID NO: 15). Consequently, the α-acetolactate synthase activity of L. lactis subsp. lactis is possible only if both of these genes are expressed.

The subject of the invention is, more especially, the fragments of the above nucleic acid coding for one or both subunits of a protein capable of possessing the enzymatic properties of α-acetolactate synthase of L. lactis subsp. lactis. More specifically, the invention relates to nucleic acids comprising one or other or both of the following segments:

the first segment corresponding to the ilvB gene (SEQ ID NO: 12), bounded by the nucleotides located at positions 8018 and 9742 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for the polypeptide of 575 amino acids (SEQ ID NO: 7) corresponding to one of the two subunits of α-acetolactate synthase of L. lactis subsp. lactis;

the second segment corresponding to the ilvN gene (SEQ ID NO: 13), bounded by the nucleotides located at positions 9738 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for the polypeptide of 158 amino acids (SEQ ID NO: 8) corresponding to the other subunit of α-acetolactate synthase of L. lactis subsp. lactis.

The invention also relates to the above nucleic acids in which the sequences are modified, provided the polypeptides encoded by these nucleic acids retain their enzymatic properties of the α-acetolactate synthase type.

Such modifications, without implied limitation, lead, for example, to variant nucleic acids which differ from the nucleic acid of the invention by the addition and/or deletion of one or more nucleotides and/or modification of one or more nucleotides.

Thus, the invention further relates to DNA sequences coding for α-acetolactate synthase of the pathway of synthesis of the branched amino acids of bacteria of the genus Lactococcus, such as L. plantarum, L. rafinolactis and L. lactis, for instance L. lactis subsp. cremoris; which DNA sequences display a strong homology with the nucleic acid sequence of ilvBN (SEQ ID NO: 14).

Advantageously, these DNA sequences are identified, and where appropriate selected, by genetic hybridization with one or more nucleic acid probes originating from the above nucleic acid sequences, under moderately stringent conditions.

As an example, a method for the identification of such a DNA sequence comprises the following steps:

extraction of the DNA from the bacterium;

digestion of this DNA with one or more restriction enzymes such as EcoRI;

transfer of the DNA fragments obtained onto a nitrocellulose membrane;

hybridization with the complete ilvBN sequence in a hybridization solution whose composition is: 6×SSC, approximately 10% formamide, 5×Denhardt, 100 ml of phosphate buffer pH 7.

The invention also relates to polypeptide sequences comprising all or part of the polypeptide sequences encoded, on the one hand by the DNA sequence bounded by the nucleotides located at positions 8018 and 9742 of SEQ ID NO: 11 and as shown in FIG. 2, and on the other hand by the DNA sequence bounded by the nucleotides located at positions 9738 and 10211 of SEQ ID NO: 11 and as shown in FIG. 2; these two polypeptides constituting the subunits of a protein possessing the enzymatic properties of α-acetolactate synthase of L. lactis subsp. lactis.

The subject of the invention is also any polypeptide sequence originating from each of the above polypeptide sequences, provided the protein formed from these polypeptides possesses enzymatic properties of the type possessed by α-acetolactate synthase.

The above polypeptides may be modified provided they retain the enzymatic properties defined above. For example, without implied limitation, polypeptides lying within the scope of the invention can differ from those defined above by the addition and/or deletion of one or more amino acids and/or modification of one or more amino acids.

A person skilled in the art has at his disposal means enabling him to identify those polypeptides of shorter sequences which lie within the field of the invention. A general means enabling him to undertake this identification consists in treating each of the above polypeptides with a protease that cleaves the polypeptides at a specific site, and then separating the fragments obtained and testing them for their enzymatic activity with respect to pyruvate.

Another means enabling regions of the polypeptides which are essential to the α-acetolactate synthase activity to be identified consists in cleaving the nucleic acids corresponding to the ilvB and ilvN genes, for example using one or more restriction enzymes, before introducing them into the cell host or the expression vector used for the production of a protein displaying α-acetolactate synthase activity. The truncated nucleic acid may thus be tested for its capacity to express effectively a product possessing α-acetolactate synthase activity or, on the contrary, no longer possessing it.

The subject of the invention is also any recombinant nucleic acid comprising at least one nucleotide sequence of the abovementioned type coding for α-acetolactate synthase, combined with at least one promoter and/or one transcription terminator recognized by the polymerases of the host into which the said recombinant nucleic acid is introduced.

The introduction of the recombinant nucleic acid may be carried out using vectors of the plasmid type capable of replicating in the host microorganism and of permitting the expression therein of the sequence coding for the enzyme, or alternatively the recombinant nucleic acid may be introduced directly into the genome by genetic engineering techniques known to a person skilled in the art.

The host microorganisms into which the recombinant nucleic acid is capable of being introduced are preferably prokaryotic cells such as Lactococcus lactis, Leuconostoc lactis, Lactobacillus lactis, Lactobacillus plantarum, but may also be eukaryotes such as yeasts.

More especially, the invention relates to all microorganisms capable of being used as leaven in an agri-foodstuffs process, in which microorganisms the recombinant nucleic acid of the invention is introduced for the purpose of obtaining an overproduction of diacetyl in an agri-foodstuffs product such as dairy product derivatives, fermented wheys, soft white cheeses, and the like.

Advantageously, the introduction of the recombinant nucleic acid of the invention into L. lactis bacteria is combined with an inhibition of the enzyme responsible in these bacteria for the conversion of acetolactate to acetoin (α-acetolactate decarboxylase) and/or of the enzyme involved in the conversion of pyruvate to lactate (lactate dehydrogenase), in order to increase the acetolactate pool leading to diacetyl.

The inhibition of these enzymes may be achieved by all chemical or biological means that enable the metabolic flux towards diacetyl production to be increased; a first biological means consists in deleting all or part of the wild-type genes coding or these enzymes; a second biological means consists in replacing the wild-type genes coding for these enzymes by mutated genes incapable of expressing the enzymes in question.

The recombinant nucleic acid of the invention may also be introduced into other microorganisms, such as Bacillus, for the production of diacetyl for non-agri-foodstuffs purposes, for example in cosmetology and as an antibacterial.

Other features of the invention will become apparent in the description which follows, which relates on the one hand to the cloning, characterization and sequencing of the region of L. lactis subsp. lactis DNA carrying the genes involved in the pathway of synthesis of the branched amino acids, and on the other hand to the introduction of a nucleic acid according to the invention, coding for a polypeptide displaying α-acetolactate synthase activity, into a cell host.

I--CLONING OF THE GENES INVOLVED IN THE SYNTHESIS OF AMINO ACIDS IN L. LACTIS SUBSP. LACTIS 1) Materials and Methods

a) Bacterial strains, plasmids and culture media

The bacterial strains, plasmids and phages used for gene cloning are listed in Table I below.

                  TABLE I                                                          ______________________________________                                                                      Bibliographic                                                                  references                                        Strains and                  or                                                plasmids  Characteristics    sources                                           ______________________________________                                         L. lactis subsp.                                                               lactis                                                                         NCDO2118  natural isolate    NCDO                                              B. subtilis                                                                    CU740     leuA5 trpC2 (SPβ)                                                                            (7)                                               CU741     leuC7 trpC2        (7)                                               CU315     leuD117 trp2 (SPβ)                                                                           (15)                                              IL2685    leuB6 trpC2 ilvA r.sup.- /m.sup.- Cm.sup.R                           MT119     leuB6 trpC2 r.sup.- /m.sup.-                                                                      (16)                                              GSY184    ilvC1 trpC2        (17)                                              IL3151    ilvD4 leuB6 r.sup.- /m.sup.+                                         1012      leuA8.sup.a metb5 r.sup.- /m.sup.+                                                                (18)                                              GSY276    ilvD4 trpC2        (17)                                              E. coli                                                                        CU518     leuA371            (19)                                              CU514     leuB401            (19)                                              CU520     leuC171            (19)                                              CUS26     leuD101            (19)                                              AB1255    tonA2 lacY1 tsx-5 supE44                                                                          (20)                                                        gal-6 λ.sup.- hisG1 rpsL8 malA1                                         xyl-7 mtl-2 ilvA201 metB1                                            FD1062    argH1 thi-1 ara-14                                                             ilvI614 ilvH612 λ.sup.-                                                 glyA18 relA1 spoT1                                                             ilVB619 bglR20 rbs-5::tn5                                            JP58      ilvG468 (ilvG.sup.+ ) thi-1                                                                       (21)                                                        galK2 λ.sup.- rpSL704 xyl-5                                   TG1       mtl-1 ilvC7 argE3 thi-1                                                                           (22)                                                        supE thi D (lac-proAB)                                                         hsdD5 F.sup.+ traD36 proAB                                                     lacI zΔ15                                                      Plasmids                                                                       pIL253                       (23)                                                        Em.sup.R, 4.9-kb                                                     pHV438                       (42)                                                        Hybrid between pBR322,                                                         the Cm.sup.R gene of pC194,                                                    thyB and the X segments                                                        of B. subtilis DNA                                                   pIL371    9.2 kb Sau3A fragment                                                          of the L. lactis                                                               chromosome in pIL253                                                 pIL373    4.5-kb Sau3A fragment                                                          of the L. lactis                                                               chromosome in pIL253                                                 pIL374    8.5-kb Sau3 fragment                                                           of the L. lactis                                                               chromosome in pIL253                                                 pIL384    13-kb Sau3A fragment                                                           of the L. lactis                                                               chromosome in pIL253                                                 pIL389    7.5-kb left-hand Sau3A                                                         fragment of pIL384 in                                                          pBS                                                                  pIL500    18.5-kb XbaI fragment                                                          of the L. lactis                                                               chromosome in pIL253                                                 pIL505    6.5-kb right-hand                                                              SmaI-Sau3A fragment of                                                         pIL384 in pBS                                                        pIL533    2.5-kb left-hand                                                               Sau3A-ExoIII fragment                                                          of pIL384                                                            pBluescript                                                                              Ap.sup.R, M13 ori, pBR322                                                                         (Strata-                                                    ori                gene)                                             ______________________________________                                    

L. lactis subsp. lactis strain IL1403 was cultured at 37° C. on M17 medium (25) in which lactose was replaced by glucose. Escherichia coli was cultured on Luria-Bertani (LB) medium or in M9 minimum medium (26) at 37° C. Bacillus subtilis was cultured on a medium at 37° C., on LB medium or in Spizizen-salt minimum medium supplemented with the appropriate amino acids. If necessary, erythromycin (5 μg/ml for L. lactis subsp. lactis or 0.3 μg/ml for B. subtilis), ampicillin (50 μg/ml for E. coli) or tetracycline (10 μg/ml for E. coli) is added to the medium.

b) Molecular cloning and DNA manipulation

The plasmids and the chromosomal DNA were prepared according to known techniques of the prior art (26, 27, 28, 29). Transformation of E. coli cells was carried out either by a standard CaCl₂ procedure (36) or by electroporation (30). Induction of competence and transformation of B. subtilis cells were carried out as described by Anagnotopoulos and Spizizen (31), with a few modifications (32). Southern blotting and DNA hybridizations were carried out as described by Maniatis et al. (26). Nucleic acid probes were prepared using [α-³² P]-dCTP and a nick translation kit in accordance with the supplier's recommendation. The other molecular techniques were carried out as described by Maniatis et al. (26).

c) Analysis of the DNA sequence

The E. coli clones used for sequencing were obtained by subcloning DNA fragments into plasmids of the pBluescript family and using exonuclease III and mung bean nuclease from the company Stratagene in order to generate a series of clones containing overlapping DNA fragments.

The DNA was then sequenced using the "Taq DyeDeoxy Terminator Cycle Sequencing" kit and the "Sequencer 370A" marketed by the company Applied Biosystems. Nucleotide and polypeptide sequences were analysed with the University of Wisconsin's "BISANCE" and "GCG" softwares. Protein sequences were aligned with a "MULTALIN" software (33).

The nucleic acid sequences were determined on both strands. Restrictively, B. subtills strains mutated in the ilvA gene were prepared by transforming competent MT119 cells with pHV438 (34) to Cm^(R). Integration of this plasmid in the chromosome by double crossing-over replaces the ilvA gene by the Cm^(R) gene (24).

A representative ilvA leuB6 r⁻ /m⁻ Cm^(R) clone is designated IL2685.

An ilvD4 leuB6 double mutant of B. subtilis was constructed by transformation, using GSY276 DNA to transform competent 1012 cells to cells which were prototrophic for methionine. The transformed cells were then tested for their requirements for isoleucine and leucine and for the absence of restriction by titration of the rho phage.

An ilvD4 leuB6 r⁻ /m⁺ clone was designated IL3151.

2) Gene Cloning

Total DNA of L. lactis subsp. lactis NCDO2118 was partially digested with the endonuclease Sau3A1. 20 μg of DNA segments of size larger than 10 kb were ligated with 10 μg of DNA of plasmid vector pIL253 cleaved with BamHI, at a final concentration of 500 μg/ml.

The ligated mixture was used to transform competent cells of the mutant B. subtilis strain leuB6 ilV4, IL3151 to isoleucin-independent cells. Four transformants were also Em^(R), and contained 18-, 14.2-, 13.5- or 9.5-kb plasmids designated pIL384, pIL371, pIL374 and pIL373, respectively.

According to a similar experiment, L. lactis subsp. lactis DNA cleaved with XbaI was cloned into pIL253 using the mutant B. subtilis ilvA strain IL2685 as recipient. The transformants were selected on a minimum medium enriched with leucine and tryptophan but lacking isoleucine. Two Em^(R) Ile⁺ clones, containing an apparently identical 23.5-kb plasmid, designated pIL500, were obtained.

3) Complementation Experiments

The cloned DNA segments were used to complement, in B. subtilis, leu and ilv mutants, and in E. coli after subcloning into pBluescript. The results relating to these studies are reported in FIG. 1.

FIG. 1 shows the structure of the DNA region carrying the genes for the enzymes involved in the pathway of synthesis of the branched amino acids in L. lactis. In this figure, the open bars represent the segments used for the complementation experiments in B. subtilis and E. Coli. These segments are carried by the plasmids indicated in the figure, which were constructed as described in Table I above. The results of the complementation experiments with the mutants listed in Table I are represented by (+) and (-), referring to growth or the absence of growth on the culture medium, lacking the corresponding amino acids. The organization of the DNA region carrying the genes involved in the pathway of synthesis of the branched amino acids (orf1, leuA, leuB, leuC, leuD, orf2, ilvD, ilvB, ilvN, ilvC, ilvA) is also shown at the bottom of FIG. 1; p1 and p2 indicate the transcription promoters and t1 the transcription terminator. The open reading frame between p1 and t1 represents the leader peptide.

In these complementation experiments, the E. coli nomenclature has been used; the leuB and leuC genes of E. coli correspond to the leuC and leuB genes, respectively, of B. subtils, and the three isoenzymes of acetolactate synthase and of acetohydroxide synthase encoded by ilvBN, ilvIH and ilvGN in E. coli correspond to a single enzyme encoded by ilvBN in B. subtilis. The leuA, leuB, leuC, ilvD and ilvA genes complement the corresponding mutations in B. subtilis ; the leuA, leuB, leuC, leuD and ilvC genes complement the corresponding mutations in E. coli.

These results are sufficient to identify nine genes involved in the pathway of biosynthesis of the branched amino acids in L. lactis subsp. lactis.

4) Nucleotide Sequences of the Genes

The nucleotide sequence of a region of 12,720 bp was determined. This sequence (SEQ ID NO: 11) is shown in FIG. 2. A computer analysis according to the method of Griskov et al. (35) discloses ten open reading frames (ORF) of size larger than 200 bp. Each ORF is preceded by a ribosome binding site, complementary to the 3' end of rRNA 163 of L. lactis subsp. lactis (36). All the ORFs begin with the codon ATG, except for the second which begins with the codon TTG; furthermore, four 72-bp repeat units are present between the second and the third ORF.

FIG. 2 shows the nucleotide sequence, and the polypeptide sequence deduced from this nucleotide sequence, of the DNA region coding for the genes involved in the pathway of synthesis of the branched amino acids of L. lactis strain NCDO2118; the numbers at the right indicate the position of the oligonucleotides; the names of the genes are indicated at the beginning of each amino acid sequence; the transcription stop codons and also the ribosome binding sites are indicated respectively by (*) and RBS; the consensus sequences "-10" and "-35" of the promoters are underlined; inverted repeat units, which can constitute "preamptors" and "terminators", are numbered (1,2) and (5, 6), respectively, and those capable of forming "antiterminators" are numbered (3and 5), (see FIG. 4 below); the arrow (→) indicates the 72-bp direct repeat unit, and the bold characters indicate the 6-bp inverted repeat units which it contains.

5) Assignment of the ORFs

The proteins deduced from the ten ORFs were compared with the GENPRO and NBRF protein databases. Significant homologies were identified for ten ORFs; nine of them correspond to the genes involved in the pathway of synthesis of the branched amino acids, as seen in Table II below.

                                      TABLE II                                     __________________________________________________________________________             %.sup.a of identical amino acids                                                                              Bibliographic                           Microorganism                                                                          LeuA                                                                               LeuB                                                                               LeuC                                                                               LeuD                                                                               IlvD                                                                              IlvB                                                                              IlvN                                                                              IlvC                                                                              IlvA                                                                              references                              __________________________________________________________________________     E. coli --.sup.b                                                                           --  --  46  42 42.sup.c                                                                          <20.sup.d                                                                         34 36 (13, 11, 12, 37)                                                   43.sup.e                                                                           37.sup.f                                                                               (38)                                                               40.sup.R                                                                          <20.sup.h                                                                               (11)                                    Salmonella                                                                             41  --  49  --  -- -- -- -- -- (39, 40)                                tryphenurium                                                                   Bacillus                                                                               --  53  --  --  -- -- -- -- 50 Genbank (41)                            subtilis                                                                       Bacillus                                                                               --  53  --  --  -- -- -- -- -- (42)                                    coagulans                                                                      Thermus --  42  --  --  -- -- -- -- -- (43)                                    aquaticus                                                                      Saccharomyces                                                                          25  46  50.sup.i                                                                           52.sup.j                                                                           -- 41 -- 33 38 (44, 45, 46, 47,                        cerevisiae                             48, 49)                                 Phycomyces                                                                             --  --  50.sup.i                                                                           49.sup.j                                                                           -- -- -- -- -- (50)                                    blakesleeanus                                                                  Mucor   --  --  49  --  -- -- -- -- -- (51)                                    circinelloides                                                                 __________________________________________________________________________      In Table II:                                                                   .sup.a indicates that the percentage was calculated using a software (52)      from the percentage identity by the size of the smallest protein;              .sup.b indicates sequences not available;                                      .sup.c indicates the comparison with IlvB nucleic acid;                        .sup.d indicates the comparison with IlvN nucleic acid;                        .sup.e indicates the comparison with IlvI nucleic acid;                        .sup.f indicates the comparison with IlvH nucleic acid;                        .sup.g indicates the comparison with IlvC nucleic acid;                        .sup.h indicates the comparison with IlvM nucleic acid;                        .sup.i Nterminal end of Leu1;                                                  .sup.j Cterminal end of Leu1.                                            

In addition, two genes detected by complementation, the ilvB gene (homologous to ilvB, ilvI and ilvG of E. coli ) and the ilvN gene (homologous to ilvH of E. coli ), for which complementation data were not obtained, were identified.

Most of the proteins of L. lactis were similar in size to their homologues. However, three exceptions were observed. Relative to the proteins of E. coli, the lactococcal proteins IlvA and IlvC lack the C-terminal amino acids 73 and 147, respectively, and IlvD displays a deletion of 36 amino acids. One of the ORFs, designated ORF2, codes for a protein not displaying any homology with the enzymes of the pathway of synthesis of the branched amino acids, but carries two regions that are conserved in a superfamily of ATP-binding proteins (53).

FIG. 3 shows the alignment of 6 ATP-binding proteins with the ORF2 sequence of L. lactis. Needleman's software was used to obtain these results (54). In FIG. 3:

the binding domains (53) are represented by NB1 and NB2;

a short sequence shared by all the members of the ATP-dependent family is indicated by the symbols (#);

the symbols (λ) indicate amino acids common to all the proteins;

the symbols (.) indicate conserved substitutions;

the amino acids shown in bold characters are present in at least five proteins;

the figures at the left indicate the position of the amino acids, and the figures in brackets indicate the size of the proteins;

the functions of the different proteins are shown in the following manner:

(¹), nodulation in Rhizobium leguminosarum (55);

(²), maltose transport in E. coli (56);

(³), glutamine transport in E. coli (57);

(⁴), glycine, betaine and proline transport in E. coli (58);

(⁵), haemolysin secretion in E. coli (59);

(⁶), cyclolysine secretion in Bordetella pertussis (60).

The organization of the sequenced segments is presented in FIG. 1. All the genes, except for the first one, upstream of LeuA (ORF1), are transcribed in the same direction. The leu and ilv genes are grouped together, and the two groups are separated by 121 bp. The leu genes are separated by less than 19 bp, except for leuB and leuC which are separated by four 72-bp direct repeats. The distance between the ilv genes is between approximately 10 and 42 bp, except for ilvB and ilvN which are separated by 9 bp.

6) Transcription Signals

The sequences conforming to the consensus units of the lactococcus promoters were identified upstream of the assemblies of leu and ilv genes (see the positions of p1 and p2 in FIG. 1). The region between p1 and leuA strongly resembles the regulatory regions of the amino acid biosynthesis operons controlled by attenuation (61). The transcript initiated at p1 can follow two paths. One to the formation of a rho-independent transcription "terminator" (as shown in FIG. 4A). In addition, the transcript carries a 51-bp message, beginning with an ATG codon and ending with a TAG codon, which determines a leader peptide of 16 amino acids, four of which are a succession of leucine and isoleucine (as shown in FIG. 4B). The ribosome sited at the Leu and Ile codons prevents formation of the termination signal and leads to transcription of the leu genes downstream. Independent transcription "terminators" were not found between the two assemblies of genes or downstream of the ilvA gene.

7) Organization of the Ilv Operon

The genes involved in the pathway of synthesis of the branched amino acids in L. lactis subsp. lactis are organized in a large assembly divided into two units grouping together the leu and ilv genes. The two units are necessary for the synthesis of leucine, whereas only the second is required for the synthesis of isoleucine and valine. A transaminase, involved in the last step of the pathway of synthesis of the branched amino acids (product of the ilvE gene in E. coli ), is not encoded by the assembly. This implies that this reaction is implemented by a non-specific transaminase, or alternatively that the corresponding gene in L. lactis subsp. lactis occurs elsewhere on the chromosome.

The leu and ilv genes are both preceded by a promoter; however, they are not separated by an independent transcription "terminator", suggesting that they form a single operon. This operon extends beyond the last biosynthesis gene, ilvA.

8) Regulation of the Ilv Operon

Sequence analysis strongly suggests that the operon is regulated by an attenuation mechanism, the mediator of which is a leucine-rich leader peptide. This peptide very closely resembles the leader peptide of the leu operon of E. coli and of S. typhimurium (62, 63), but differs from that of the ilvBN and ilvGMEDA operons of E. coli.

FIG. 4 shows the secondary structures of the leader transcript capable of participating as a mediator in transcription attenuation:

at A, the termination topology is shown; units (1) and (2) are paired, and units (5) and (6) form a transcription terminator;

at B, the antitermination topology is shown; units (3) and (4) are paired, and mask units (5) and (6).

In FIG. 4, the nucleotides in bold characters indicate the sequence coding for the leader peptide, and the nucleotides in bold italics indicate the ribosome binding sites of the leader peptide and the leuA gene. Stalling of the ribosome at the successive Leu and Ile residues could mask unit (1) and favour this topology.

Current models propose that the strength of the attenuation is dependent on the tRNA^(lcu) load during translation of the leader peptide. The presence of rare codons increases the response to leucine starvation by increasing the duration of ribosome stalling. In E. coli, the codon corresponding to the four leucine residues present in the leader peptide is CUA, which corresponds to only 2% of the leucine codons used for the proteins of this organism (64). In contrast, the leucine codon UUC, encountered three times in the leader transcript of the pathway of synthesis of the branched amino acids in L. lactis, corresponds to 24% of the leucine codons in the proteins of L. lactis (65). The isoleucine codon is also present in the leader peptide of L. lactis, following very closely the Leu codons, which is not the case in E. coli, and may hence affect the response of the operon to an arrest of the pathway of synthesis of the branched amino acids. Other studies are necessary to determine whether the model proposed for regulation in E. coli may be applied directly to L. lactis.

9) The Open Reading Frame ORF2

ORF2, inasmuch as it corresponds to a product belonging to the superfamily of ATP-binding proteins, is not a biosynthesis gene in the pathway of synthesis of the branched amino acids. The proteins of this superfamily are encountered both in prokaryotes and in eukaryotes, and share a similar base organization (66, 67). In prokaryotes, most of the members of this superfamily are components of the transport systems involving periplasmic binding proteins. These genes are generally cotranscribed with other genes (67), and have never been found in the biosynthesis operon. Other studies are necessary to establish the function of ORF2, but the latter is doubtless involved in the transport of the chain of synthesis of the branched amino acids, or in regulation of the genes of this pathway.

Isoleucine, leucine and valine represent 20% of the amino acids of L. lactis (68), whereas tryptophan and histidine, two other amino acids whose pathways of biosynthesis in L. lactis have been studied, represent less than 2%. This suggests that a fine regulation of the expression of the genes of the pathway of synthesis of the branched amino acids is necessary.

II--INTRODUCTION OF A NUCLEIC ACID CODING FOR AN α-ACETOLACTATE SYNTHASE INTO A HOST

The nucleic acid consisting of the DNA sequence bounded by the nucleotides located at positions 7977 and 10246 of SEQ ID NO: 11 and as shown in FIG. 2, and coding for the IlvB and IlvN polypeptides, is amplified by the Taq polymerase chain reaction from the DNA of FIG. 2, and using two oligo-nucleotides located upstream and downstream of the nucleic acid sequence coding for IlvBN.

The following two oligonucleotides were used:

CTAGTGAAGGTTGCGTTACA (SEQ ID No: 15)

TGCCATTTTTGTTTCCTCTA (SEQ ID No: 16)

After the polymerase chain reaction, the product of the reaction is cloned into an expression vector, such as pIL252, pIL253, pWV01, containing a promoter such as the promoter of the lactose operon or of the tryptophan operon, and where appropriate a terminator.

After cloning of the amplified segment in the appropriate orientation, the plasmid obtained is introduced by transformation into a strain of L. lactis, such as IL1403 or MG1363, which expresses IlvBN at a high level. This results in diversion of a part of the pyruvate pool obtained after glycolysis towards α-acetolactate, a precursor of diacetyl.

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    __________________________________________________________________________     SEQUENCE LISTING                                                               __________________________________________________________________________     LOCUS    LACLEUILV                                                                               12720 bp ds-DNA                                                                          BCT                                                  02-NOV-1992                                                                  DEFINITION                                                                              Lactococcus lactis operon (leuA, leuB, leuC,                          leuD, ilvD, ilvB,                                                                       ilvN, ilvC, ilvA) genes complete cds; complete                        ORF2.                                                                          ACCESSION                                                                               M90761                                                                KEYWORDS  ilvA gene; ilvB gene; ilvC gene; ilvD gene;                          ilvN gene; leuA gene;                                                                   leuB gene; leuC gene; leuD gene; operon.                              SOURCE   Lactococcus lactis (library: NCDO2118) DNA.                            ORGANISM                                                                               Lactococcus lactis                                                             Prokaryotae; Firmicutes; Firmibacteria;                               Gram-positive cocci;                                                                    Streptococcaceae.                                                     REFERENCE                                                                               1 (bases 1 to 12720)                                                   AUTHORS   Godon, J.-J.J.-J., Chopin, M.-C.M. and                              Ehrlich, D. S.                                                                  TITLE   Branched-chain amino acid biosythesis genes in                        Lactococcus lactis                                                                      subup. lactis.                                                         JOURNAL J. Bacteriol. 174, 6580-6589 (1992)                                    STANDARD                                                                               full automatic                                                        FEATURES    Location/Qualifiers                                                  attenuator                                                                               260. .450                                                                      /note="putative"                                                     repeat.sub.-- region                                                                     3039. .3327                                                                    /function="unknown"                                                            /rpt.sub.-- type=DIRECT                                                        /note="putative"                                                     CDS       450. .1991                                                                     /EC.sub.-- number="4.1.3.12"                                                   /gene="leuA"                                                                   /citation=[1]                                                                  /note="putative"                                                               /codon.sub.-- start=1                                                                    / t r a n s l a -                                        tion="MRKIEFFDTSLRDGEQTPGVSFSISEKVTIAKQLEKWRISVIEA                             GFSAASPDSFEAVKQIADSLNDTAVTALARCVISDIDKAVEAVKGAKYPQIHVFIATS                     PIHMKYKLKISPEEVLKNIDKCVRYARERVEVVEFSPEDATRTELNFLLEAVQTAVDA                     GATYINIPDTVGYTTPEEYGKIFKFLIDNTKSDREIIFSPHCHDDLGMAVANSLAAIK                     AGAGRVEGTVNGIGERAGNAALEEIAVALHIRKDFYQAQSPLKLSETAATAELISQFS                     GIAIPKNKAIVGANAFAHESGIHQDGVLKNAETYEIITPELVGIKHNSLPLGKLSGRH                     AFSEKLTELNIAYDDESLAILFEKFKKLADKKKEITDADIHALFTGETVKNLAGFILD                     NVQIDGHKALVQLKNQEEEIYVSQGEGSGSVDAIFKAIDKVFNHQLKLISYSVDAVTD                     GIDAQATTLVSVENLSTGTIFNAKGVDYDVLKGSAIAYMNANVLVQKENLQGKVEQIS                                 AHDGI"                                                             CDS         2003. .3040                                                                    /gene="leuB"                                                                   /note="putative"                                                               /codon.sub.-- start=1                                              /translation="LSKKIVTLAGDGIGPEIMSAGLSVLKAVSKKIDFEYELEAKDFG                     GIAIDKHGHPLPEETLQAVKNADAILLAAIGHPKYNNAKVRPEQGLLALRKELGLYAN                     VRPLKIYPALKKLSPIRNVENVDFLVIRELTGGIYFGQHELADDKARDVNDYSADEIR                     RILHFAFKSAQSRPRKLLTSVDKQNVLATSKLWRKMADEIADEYPDVRLEHQLVDSCA                     MLLITNPQQFDVIVTENLFGDILSDEASSLAGSLGVMPSSSHGFNGLALYEPIHGSAP                     DIAGKGIANPVSMILSIAMMLRESFGQEDGAAMIEKAVTQTFTDGILTKDLGGTATTK                                 EXTEAILKNCQ"                                                       CDS         3403. .4785                                                                    /gene="leuC"                                                                   /note="putative"                                                               /codon.sub.-- start=1                                              /translation="MSGKTIFDKLWDQHVIAGNEGEPQLLYIDLHVIHEVTSPQAFQG                     LREAGRRVRRKDLTYGTLDHNVPTQNIFNIQDLISKKQIDTFTKNVKEFDVPAETHGG                     KGQGIVHMVAPESGRTQPGKTIVCGDSHTATNGAFGAIAFGIGTSEVEHVLATQTIWQ                     VKPKRMKIEFQGHPQKGIYSKDFILALIAKYGVDAGVGYAVEYSGDAISDLSMEERMT                     ICNMSIEFGAKIGLMNPDEKTYDYVKGREHAPKNFDEAVSKWEKLVSDSDAQYDKILS                     LDVSQLKPMVTWGTNPGMGLEFGEKFPEINNDLNYERAYQYMDLKPGQTASDIDLGYI                     FIGSCTNARLGDLEEAAKIIGDRHIADGLTGIVVPGSRPVKEAAEAQGLDKIFKEAGF                     EWREPGCSACLGMNPDQIPEYVHCASTSNRNFEGRQGHNARTHLCSPAMAAAAAIAGK                                 FVDVRMLVTD"                                                        CDS         4805. .5380                                                                    /gene="leuD"                                                                   /note="putative"                                                               /codon.sub.-- start=1                                              /translation="MEKFTIYKGTSVPVMNDNIDTDQIIPKQFLKAIDKKGFGKNLFY                     EWRYLKDYDENPDFILNAPKYKKASLLISGDNFGSGSSREHAAWALSDYGFRAIIAGS                     YSDIFYNNALKNGLLPIKQPREVLNQLTKLSSQEEITIDLPHQLIITSLGDFHFEIDP                                 IWKDKLINGLDDIGITLQYEEAISAYEQKNQ"                                   CDS         5394. .6173                                                                    /function="unknown"                                                            /note="ORF2; putative"                                                         /codon.sub.-- start=1                                              /translation="MTIINLKNVNLTRNKKEILKDITWKVNPGENWVILGLNGSGKSS                     LLKLILAEEWKTSGEITVLNTQFRNGEIPKLRKRISVVGSFIAERFQPNIKAENLVYT                     GKFNSSMLYKPYTDQELDEARQLLRQMGAKSLIGRNYASLSQGEKQVLLIARSLILKP                     ELLILDEATNGLDLFAKEKLLKQLQQINQLKTAPTLIYISHHPDEITDIFTHLLLLRE                     GKVIQSGKKENLLNEKILTDFYQEKVEVHRFEQKYFVIPAN"                                     CDS         6295. .8007                                                                    /gene="ilvD"                                                                   /note="putative"                                                               /codon.sub.-- start=1                                              /translation="MEFKYNGKVESVELNKYSKTLTPRSTQPATQAMYYGIGFKDEDF                     KKAQVGIVSMDWDGNPCNMHLGTLGSKIKSSVNQTDGLIGLQFHTIGVSDGIANGKLG                     MRYSLVSREVIADSIETNAGAEYYDAIVAIPGCDKNMPGSIIGMARLNRPSIMVYGGT                     IEHGEYKGEKLNIVSAFEALGQKITGNISDEDYHGVICNAIPGQGACGGMYTANTLAA                     AIETLGMSLPYSSSNPAVSQEKQEECDDIGLAIKNLLEKDIKPSDIMTKEAFENAITI                     VMVLGGSTNAVLHIIAMANAIGVEITQDDFQRISDIIPVLGDFKPSGKYMMEDLHKIG                     GLPAVLKYLLKEGKLHGDCLTVTGKTLAENVETALDLDFDSQDIMRPLKNPIKATGHL                     QILYGNLAQGGSVAKISGKEGEFFKGTARVFDGEQHFIDGIESGRLHAGDVAVIRNIG                     PVGGPGMPEMLKPTSALIGAGLGKSCALITDGRFSGGTHGFVVGHIVPEAVEGGLIGL                     VEDDDIIEIDAVNNSISLKVSNEEIAKRRANYQKPTPKATRGVLAKFAKLTRPASEGC                                 VTDL"                                                              CDS         8018. .9745                                                                    /gene="ilvB"                                                                   /note="putative"                                                               /condon.sub.-- start=1                                             /translation="MKKIKLEKPTSGSQLVLQTLKELGVEIIFGYPGGAMLPLYDAIH                     NFEGIQHILARHEQGATHEAEGYAKSSGKVGVVVVTSGPGATNAVTGIADAYLDSVPL                     LVFTGQVGRQSIGKDAFQEADTVGITAPITKYNYQIRETADIPRIVTEAYYLARTGRP                     GPVEIDLPKDVSTLEVTEINDPSLNLPHYHESEKATDEQLQELLTELSVSKKPVIIAG                     GGINYSGSVDIFRAFVEKYQIPVVSTLLGLGTLPISHELQLGMAGMHGSYAANMALVE                     ADYIINLGSRFDDRVVSNPAKFAKNAVVAHIDIDAAELGKIVKTDIPILSDLKAALSR                     LLQLNKVRTDFNDWIKTVIENKEKAPFTYEPQNHDIRPQETIKLIGEYTQGDAIIVTD                     VGQHQMWVAQYYPYKNARQLITSGGMGTMGFGIPAAIGAKLAQPNKNVIVFVGDGGFQ                     MTNQELALLNGYGIAIKVVLINNHSLGMVRQWQESFYEERRSQSVFDVEPNFQLLAEA                     YGIKHVKLDNPKTLADDLKIITEDEPMLIEVLISKSEHVLPMIPAGLHNDEMIGLHFT                                 DKNEEIDNA"                                                         CDS         9738. .10214                                                                   /gene="ilvN"                                                                   /note="putative"                                                               /condon.sub.-- start=1                                             /translation="MRRMIIAKLHNVTGIMNRFTAVLNRRQVNILSITAGVTESQDLT                     HTTFVIEVDHLDEVEQIIKQLNRLIDVIEVADITDFPHVEREVVLIKVSAPPTIRAEI                     FTMIEPFRVNVVDVNLENVTIQLTGDSAKIEALIEVVSPYGILNMARTGSAGFERG"                      CDS         10260. .11294                                                                  /gene="ilvC"                                                                   /note="putative"                                                               /condon.sub.-- start=1                                             /translation="MAVTMYYEDDVEVSALAGKQIAVIGYGSQGHAHAQNLRDSGHNV                     IIGVRHGKSFDKAKEDGFETFEVGEAVAKADVIMVLAPDELQQSIYEEDIKPNLKAGS                     ALGFAHGFNIHFGYIKVPEDVDVFMVAPKAPGHLVRRTYTEGFGTPALFVSHQNASGH                     AREIAMDWAKGIGCARVGIIETTFKEETEEDLFGEQAVLCGGLTALVEAGFETLTEAG                     YAGELAYFEVLHEMKLIVDLMYEGGFTKMRQSISNTAEFGDYVTGPRIITDEVKKNMK                     LVLADIQSGKFAQDFVDDFKAGRPKLIAYREAAKNLEIEKIGAEHVKQCHSHNLVMTM                                 PLKSISNFSY"                                                        CDS         11337. .12662                                                                  /gene="ilvA"                                                                   /note="putative"                                                               /condon.sub.-- start=1                                             /translation="MISAKEVEDAYDLLKAVVTKTPLQLDPYLSNKYQANIYLKEVVT                     KTPLQLDPYLSNKYQANIYLKEENLQKVRSFKLRGAYYSISKLSDEQRSKGVVCASAG                     NHAQGVAFAANQLNISATIFMPVTTPNQKISQVKFFGESHVTIRLIGDTFDESARAAK                     AFSQDNDKPFIDPFDDENVIAGQGTVALEIFAQAKKQGISLDKIFVQIGGGGLIAGIT                     AYSKERYPQTEIIGVEAKGATSMKAAYSAGQPVTLEHIDKFADGIAVATVGQKTYQLI                     NDKVKQLLAVDEGLISQTILELYSKLGIVAEPAGATSVAALELIKDEIKGKNIVCIIS                     GGNNDISRMQEIEERALVYEGLKHYFVINFPQRPGSLRTFVSDILGPNDDITRFEYIK                     RADKGKGPCLVGILLSDASDYDSLINRIERFDNRYVNLRGNDSLYELLV"                             BASE COUNT   4254 a   2046 c   2621 g   3799 t                                 ORIGIN                                                                         M90761   Length: 12720   April 6, 1993   18:44   Type: N                       Check: 7819 . .                                                                   1      TAAAACTCGA TAATCTTGAG TCATAATTTC TCCTTAATCT                          TATTAGTACA                                                                         51    TTAGAATCCA TTATAATTTA ATCATTTTAT GTCTACCTAA                          AGCAACAAAA                                                                        101    TTGCTTGTAT ATTTTCTAAC AAGCTTAATT ATGTGGATTT                          AATTGAATAT                                                                        151    TAAAGGGAGA AGTTGTAATC TATTTGTTGT TAAATTCTTG                          TTAATACAAA                                                                        201    TAAATTTATT AAATATTATT ATTTTATTGA CAATTTAAAA                          TATTAAGAGT                                                                        251    ATTATAATGT AAATTAACAA AAAAAAGAGG AACTTGAAAT                          GACATACACA                                                                        301    CAATTTTCAT TGTTGTTGAT CAAGGTGGAC CTACATTAGC                          TTTTTTGGCT                                                                        351    AAAATATGTG GGTCCTGTTT GGCGATAGTC ATTTCGAGGA                          CCGAGAGACG                                                                        401    TCCTCACGGG CGTCTTTTTT GTTTCTTAAT AAAAAATAGA                          GGTAATATTA                                                                        451    TGCGAAAAAT TGAATTCTTT GACACAAGTT TGAGAGATGG                          CGAACAGACA                                                                        501    CCGGGCGTTA GTTTCTCCAT TTCAGAAAAA GTAACGATTG                          CTAAACAACT                                                                        551    GGAAAAATGG AGGATTTCTG TCATAGAGGC TGGTTTTTCT                          GCGGCAAGTC                                                                        601    CAGATAGTTT TGAAGCAGTA AAGCAAATTG CTGATTCTTT                          GAATGATACG                                                                        651    GCTGTCACTG CATTAGCTCG CTGTGTTATT TCAGATATCG                          ATAAAGCGGT                                                                        701    TGAAGCGGTA AAGGGGGCTA AATATCCGCA AATTCATGTT                          TTCATTGCAA                                                                        751    CTTCACCTAT TCACATGAAA TATAAACTTA AAATCAGTCC                          CGAAGAAGTT                                                                        801    TTGAAAAATA TTGATAAGTG TGTGAGATAC GCACGTGAAC                          GGGTCGAGGT                                                                        851    TGTTGAGTTT TCTCCAGAGG ATGCAACAAG AACGGAGTTG                          AATTTTCTTT                                                                        901    TAGAGGCTGT TCAAACGGCT GTCGATGCTG GAGCAACTTA                          TATTAATATT                                                                        951    CCTGACACTG TCGGTTATAC GACACCAGAA GAATATGGAA                          AAATTTTTAA                                                                        1001   ATTTTTGATT GATAATACTA AGTCTGACCG AGAAATTATT                          TTTAGTCCAC                                                                        1051   ATTGTCATGA TGATTTAGGA ATGGCTGTAG CTAATTCATT                          AGCTGCAATT                                                                        1101   AAAGCTGGGG CTGGGAGAGT TGAAGGAACT GTCAATGGTA                          TTGGAGAGCG                                                                        1151   AGCTGGGAAT GCTGCTCTTG AAGAAATTGC TGTGGCACTA                          CATATTCGTA                                                                        1201   AAGATTTTTA TCAGGCACAA AGTCCTTTAA AACTTTCAGA                          AACTGCTGCA                                                                        1251   ACGGCAGAAC TAATTTCACA ATTTTCAGGA ATTGCTATTC                          CAAAAAATAA                                                                        1301   AGCAATTGTT GGTGCTAATG CTTTTGCACA CGAATCAGGA                          ATTCATCAAG                                                                        1351   ATGGTGTCCT TAAAAATGCT GAAACTTATG AAATTATTAC                          ACCAGAACTT                                                                        1401   GTCGGAATAA AGCATAATTC GTTGCCTTTA CGTAAACTTT                          CTGGTCGTCA                                                                        1451   TGCTTTTAGT GAAAAATTGA CGGAACTTAA TATTGCTTAT                          GACGATGAAA                                                                        1501   GTCTTGCAAT TTTATTTGAA AAATTTAAAA AATTAGCTGA                          CAAGAAAAAA                                                                        1551   GAAATTACTG ACGCAGATAT TCATGCCTTG TTTACAGGAG                          AAACGGTAAA                                                                        1601   AAATCTAGCT GGATTTATAC TTGATAATGT TCAAATTGAT                          GGGCACAAGG                                                                        1651   CATTGGTGCA ACTAAAAAAT CAAGAAGAGG AAATTTATGT                          TAGCCAAGGA                                                                        1701   GAGGGGTCAG GTTCAGTGGA TGCAATTTTT AAAGCTATTG                          ATAAAGTCTT                                                                        1751   TAATCATCAA CTAAAATTAA TTTCCTATTC AGTTGATGCT                          GTAACTGATG                                                                        1801   GAATTGATGC ACAAGCAACG ACTTTGGTTT CTGTTGAAAA                          TCTATCTACA                                                                        1851   GGCACTATAT TTAATGCTAA AGGTGTTGAT TATGATGTAT                          TGAAAGGAAG                                                                        1901   CGCCATTGCT TACATGAACG CTAATGTTTT AGTTCAAAAA                          GAAAATTTAC                                                                        1951   AAGGAAAGGT TGAACAAATT TCAGCTCATG ATGGAATTTA                          AGGTGAAAAA                                                                        2001   TATTGTCTAA AAAAATTGTG ACACTTGCGG GAGATGGAAT                          TGGGCCAGAA                                                                        2051   ATTATGTCAG CTGGTTTAAG TGTTTTAAAA GCTGTCAGTA                          AAAAAATTGA                                                                        2101   TTTTGAGTAT GAATTAGAAG CTAAAGATTT TGGAGGAATT                          GCAATTGATA                                                                        2151   AGCATGGTCA TCCTTTACCA GAAGAAACTT TGCAAGCAGT                          TAAAAATGCT                                                                        2201   GACGCAATCT TGCTCGCTGC AATTGGTCAT CCTAAATACA                          ACAATGCAAA                                                                        2251   AGTTAGACCA GAACAAGGGC TACTTGCTTT ACGAAAAGAA                          TTAGGACTGT                                                                        2301   ATGCTAATGT TCGTCCATTA AAAATTTATC CGGCTCTAAA                          AAAACTTTCT                                                                        2351   CCCATACGAA ATGTTGAAAA TGTTGATTTC CTAGTGATTC                          GCGAACTTAC                                                                        2401   AGGGGGAATC TATTTCGGTC AGCATGAATT GGCAGATGAT                          AAAGCACGAG                                                                        2451   ATGTCAATGA TTATTCTGCT GATGAAATAA GGAGAATTCT                          TCATTTTGCT                                                                        2501   TTCAAAAGTG CTCAAAGTCG GCCCAGAAAA TTACTGACTT                          CGGTTGATAA                                                                        2551   ACAAAATGTT CTTGCAACTT CTAAATTATG GCGAAAAATG                          GCTGATGAAA                                                                        2601   TTGCTGACGA ATATCCTGAT GTACGATTAG AGCACCAATT                          GGTCGATTCT                                                                        2651   TGTGCGATGT TACTGATTAC TAATCCGCAA CAATTTGATG                          TGATAGTCAC                                                                        2701   TGAAAATCTA TTTGGTGATA TTCTCTCTGA TGAAGCAAGT                          AGTTTGGCCG                                                                        2751   GTAGCTTAGG AGTGATGCCT TCGAGTTCGC ATGGATTTAA                          CGGTTTAGCA                                                                        2801   CTCTATGAGC CAATTCATGG TTCGGCACCA GATATTGCAG                          GAAAAGGAAT                                                                        2851   TGCGAACCCT GTTTCGATGA TTCTATCAAT TGCCATGATG                          CTAAGAGAAT                                                                        2901   CTTTTGGGCA AGAAGATGGG GCTGCGATGA TTGAAAAAGC                          CGTAACCCAA                                                                        2951   ACTTTTACTG ACGGAATTTT GACTAAAGAT TTAGGTGGGA                          CTGCAACAAC                                                                        3001   TAAAGAAATG ACAGAAGCAA TCCTGAAAAA TTGTCAGTAA                          AATGCGATTG                                                                        3051   AATAGTGAGC ATTTTAGTTG TAGATAAAAG AACCGTCAGC                          ATAGCTGACA                                                                        3101   ATTCTGTCAG TAAATGCGAT TGAATAGTGA GCATTTTAGT                          TGTAGATAAA                                                                        3151   AGAACCGTCA GCATAGCTGA CAATTCTGTC AGTAATTGCG                          ATTGAATAGT                                                                        3201   GAGCATTTTA GTTGTAGATA AAAGAACCGT CAGCATAGCT                          GACAATTCTG                                                                        3251   TCAGTAATTG CGATTGAATA GTGAGCATTT TAGTTGTAGA                          TAAAAGAACT                                                                        3301   ATCAGCGTAA CTGACAATTC TGTCAGTAAA TATTACTGAC                          AAAAAGTACA                                                                        3351   AAATTACTGA CAGAATTTGT CAGAATAAAT TTTTAAAAAA                          GGAAATAAAA                                                                        3401   AAATGTCAGG TAAAACAATA TTTGATAAAC TTTGGGATCA                          GCATGTGATT                                                                        3451   GCTGGAAATG AGGGAGAACC TCAACTGCTT TATATTGACC                          TTCATGTTAT                                                                        3501   TCATGAGGTT ACGAGTCCGC AAGCATTTCA GGGCTTACGT                          GAAGCAGGAC                                                                        3551   GTCGTGTTCG GAGAAAAGAT TTGACATACG GAACTCTTGA                          CCACAATGTT                                                                        3601   CCAACACAAA ATATTTTTAA TATTCAAGAT TTGATTTCTA                          AAAAACAAAT                                                                        3651   TGATACTTTT ACTAAAAATG TCAAAGAATT TGATGTTCCA                          GCGGAGACTC                                                                        3701   ATGGTGGAAA AGGACAAGGA ATTGTTCACA TGGTAGCACC                          TGAATCTGGC                                                                        3751   AGAACTCAAC CGGGAAAAAC AATTGTTTGT GGCGATAGTC                          ATACCGCAAC                                                                        3801   AAATGGAGCA TTTGGTGCAA TTGCTTTTGG AATTGGTACA                          AGTGAAGTTG                                                                        3851   AACATGTTCT TGCAACTCAA ACCATTTGGC AAGTTAAACC                          CAAGCGTATG                                                                        3901   AAAATTGAAT TTCAAGGTCA TCCACAAAAA GGAATTTATA                          GCAAAGACTT                                                                        3951   TATCCTCGCA TTAATTGCTA AATATGGTGT GGATGCAGGT                          GTAGGTTATG                                                                        4001   CGGTTGAATA TAGTGGGGAT GCTATCAGTG ATTTAAGCAT                          GGAAGAACGG                                                                        4051   ATGACAATCT GTAACATGTC AATTGAATTT GGGGCAAAAA                          TTGGCCTGAT                                                                        4101   GAATCCTGAT GAAAAAACTT ATGACTATGT CAAAGGGCGT                          GAACATGCAC                                                                        4151   CTAAAAACTT TGATGAAGCT GTCAGTAAAT GGGAAAAACT                          TGTCAGTGAT                                                                        4201   TCTGATGCAC AATACGATAA GATTTTAAGT CTTGATGTCA                          GCCAGTTGAA                                                                        4251   ACCAATGGTG ACATGGGGAA CAAATCCCGG AATGGGCCTA                          GAATTTGGCG                                                                        4301   AAAAGTTTCC GGAAATTAAC AATGATTTGA ATTATGAACG                          TGCTTATCAG                                                                        4351   TACATGGATT TAAAGCCAGG CCAAACCGCT TCTGACATAG                          ATTTAGGCTA                                                                        4401   TATTTTCATT GGTTCTTGTA CGAATGCTAG ACTTGGTGAT                          TTAGAAGAAG                                                                        4451   CTGCAAAAAT TATTGGAGAC AGACATATTG CTGATGGACT                          GACAGGAATT                                                                        4501   GTCGTCCCTG GAAGCAGACC TGTGAAAGAA GCGGCTGAAG                          CACAAGGGCT                                                                        4551   TGATAAAATT TTTAAAGAAG CTGGTTTTGA ATGGCGGGAA                          CCGGGTTGCT                                                                        4601   CAGCCTGTCT TGGAATGAAT CCTGACCAAA TTCCAGAATA                          CGTTCATTGT                                                                        4651   GCTTCAACCT CTAATCGAAA TTTTGAAGGT CGTCAAGGAC                          ATAATGCAAG                                                                        4701   AACGCACCTG TGCTCTCCAG CTATGGCTGC TGCCGCCGCA                          ATCGCTGGTA                                                                        4751   AATTTGTAGA TGTTAGAATG CTCGTAACAG ATTAGTCTGT                          AGAAAGAAAA                                                                        4801   AAAGATGGAA AAATTCACGA TTTACAAAGG GACAAGTGTT                          CCAGTCATGA                                                                        4851   ACGATAATAT TGACACAGAC CAAATTATTC CTAAACAATT                          TTTGAAAGCA                                                                        4901   ATCGATAAAA AGGGCTTTGG GAAAAATTTA TTTTATGAAT                          GGCGTTATCT                                                                        4951   TAAAGATTAC GATGAGAATC CTGATTTTAT TTTGAATGCT                          CCAAAATACA                                                                        5001   AAAAAGCTTC TCTGTTAATT TCAGGAGATA ATTTTGGTTC                          GGGTTCTTCA                                                                        5051   AGAGAACATG CGGCATGGGC CTTATCAGAT TACGGCTTTC                          GGGCAATTAT                                                                        5101   TGCTGGCTCT TACTCAGATA TTTTTTATAA TAATGCTTTA                          AAAAATGGCT                                                                        5151   TGTTACCAAT TAAACAACCA AGAGAAGTTC TAAATCAACT                          GACAAAACTG                                                                        5201   TCAAGTCAAG AAGAAATTAC AATTGATTTA CCCCATCAGC                          TAATCATCAC                                                                        5251   AAGCCTTGGT GACTTTCATT TTGAGATTGA CCCCATTTGG                          AAAGACAAAT                                                                        5301   TAATTAATGG CTTAGATGAT ATTGGAATAA CTTTGCAATA                          TGAAGAAGCA                                                                        5351   ATCTCAGCTT ACGAACAAAA AAATCAATAA GAGCGAGCCT                          AAAATGACAA                                                                        5401   TTATTAATTT AAAGAATGTA AATCTTACTC GAAATAAAAA                          AGAAATTCTT                                                                        5451   AAAGATATTA CTTGGAAAGT AAATCCCGGC GAAAATTGGG                          TTATTCTGGG                                                                        5501   CCTCAACGGC TCTGGAAAAT CAAGTCTTTT GAAATTGATT                          TTAGCAGAAG                                                                        5551   AATGGAAAAC TTCTGGTGAA ATCACTGTTT TAAATACTCA                          ATTTAGAAAT                                                                        5601   GGAGAAATTC CTAAGTTGAG AAAAAGAATC AGCGTAGTTG                          GCTCATTTAT                                                                        5651   TGCTGAAAGA TTTCAACCAA ATATTAAGGC TGAAAACCTT                          GTTTATACTG                                                                        5701   GGAAATTTAA TTCGAGCATG CTCTATAAAC CCTACACAGA                          TCAGGAACTT                                                                        5751   GATGAGGCCC GTCAGCTTTT AAGACAAATG GGCGCAAAAT                          CACTTATTGG                                                                        5801   CCGAAATTAT GCCAGCCTTT CTCAAGGGGA AAAGCAAGTT                          CTTCTTATTG                                                                        5851   CTAGGAGCTT AATTTTAAAG CCTGAGCTTT TAATTTTGGA                          CGAAGCAACG                                                                        5901   AACGGTTTAG ATTTATTTGC TAAAGAAAAA TTATTAAAGC                          AACTGCAGCA                                                                        5951   GATTAATCAA TTAAAAACCG CACCAACACT AATTTATATT                          TCTCATCATC                                                                        6001   CCGATGAAAT CACTGATATT TTTACTCACC TTTTACTTTT                          AAGAGAAGGA                                                                        6051   AAAGTGATTC AATCAGGGAA AAAAGAAAAC TTATTAAATG                          AAAAGATACT                                                                        6101   TACTGATTTT TATCAAGAAA AAGTAGAAGT TCACCGTTTT                          GAGCAGAAAT                                                                        6151   ATTTTGTAAT TCCTGCTAAC TGAGAAAGGA AAGCAAAAGT                          ATTTTATATA                                                                        6201   CTATATAGAA TATTCTGACA GATTATTGTA TTTTCATTTT                          TTTAGTGATA                                                                        6251   AAATAGCTCT ATGTAAATTT ACGGGGAGGT CAAAAAGATA                          ACATATGGAA                                                                        6301   TTCAAATATA ACGGAAAAGT TGAATCAGTG GAACTCAATA                          AATATTCTAA                                                                        6351   GACATTGACT CCAAGATCAA CACAACCAGC GACTCAAGCG                          ATGTACTACG                                                                        6401   GCATTGGTTT TAAAGATGAG GATTTCAAAA AAGCTCAGGT                          CGGAATCGTC                                                                        6451   AGCATGGATT GGGACGGAAA TCCATGTAAT ATGCACTTGG                          GAACACTTGG                                                                        6501   GAGTAAAATC AAAAGTTCTG TCAACCAAAC TGACGGATTG                          ATTGGACTTC                                                                        6551   AATTTCATAC TATTGGAGTT TCTGATGGAA TTGCTAACGG                          AAAGCTTGGC                                                                        6601   ATGAGATATT CTTTGGTCAG TCGTGAAGTT ATTGCTGACA                          GCATCGAAAC                                                                        6651   CAACGCTGGC GCAGAATATT ATGATGCCAT CGTTGCCATT                          CCCGGTTGTG                                                                        6701   ATAAAAATAT GCCCGGGTCA ATTATCGGAA TGGCTCGCTT                          AAATCGTCCG                                                                        6751   TCAATTATGG TCTATGGTGG AACGATTGAA CATGGCGAAT                          ATAAAGGTGA                                                                        6801   AAAATTAAAT ATTGTTTCGG CCTTTGAAGC TCTGGGGCAA                          AAAATCACTG                                                                        6851   GAAATATTTC TGATGAAGAT TATCATGGCG TTATTTGCAA                          TGCCATTCCA                                                                        6901   GGACAAGGTG CTTGCGGAGG AATGTACACT GCCAATACCC                          TGGCTGCTGC                                                                        6951   TATTGAAACT TTGGGAATGA GTTTACCTTA TTCCTCTTCC                          AATCCAGCAG                                                                        7001   TCAGTCAAGA AAAACAAGAA GAGTGTGATG ACATTGGTTT                          AGCCATCAAA                                                                        7051   AATTTATTAG AAAAAGATAT TAAACCAAGT GATATCATGA                          CCAAAGAAGC                                                                        7101   TTTTGAAAAT GCCATAACAA TTGTCATGGT CCTTGGAGGC                          TCAACCAATG                                                                        7151   CTGTGCTTCA TATCATTGCA ATGGCAAATG CCATTGGTGT                          AGAAATTACG                                                                        7201   CAAGATGATT TCCAACGTAT TTCAGATATT ATCCCTGTTC                          TTGGCGATTT                                                                        7251   CAAACCGAGC GGAAAATATA TGATGGAAGA TCTGCACAAA                          ATTGGTGGCC                                                                        7301   TTCCTGCTGT TTTGAAATAC CTACTTAAAG AAGGAAAACT                          TCACGGTGAT                                                                        7351   TGTTTGACCG TCACAGGTAA AACTTTGGCT GAAAATGTTG                          AAACAGCATT                                                                        7401   AGATTTGGAC TTTGACAGTC AAGATATTAT GCGACCACTA                          AAAAATCCAA                                                                        7451   TTAAAGCTAC TGGACATTTA CAAATTTTGT ACGGTAATCT                          TGCCCAAGGG                                                                        7501   GGTTCTGTTG CAAAAATTTC TGGTAAAGAA GGCGAATTTT                          TCAAAGGAAC                                                                        7551   AGCTCGTGTT TTTGACGGAG AACAACACTT TATCGATGGC                          ATTGAGTCTG                                                                        7601   GCCGATTGCA TGCCGGTGAT GTTGCGGTCA TTAGAAATAT                          TGGCCCAGTC                                                                        7651   GGAGGTCCGG GAATGCCAGA GATGTTAAAA CCAACCTCAG                          CATTAATTGG                                                                        7701   AGCAGGACTT GGAAAATCTT GTGCCCTAAT TACTGACGGA                          AGATTTTCTG                                                                        7751   GTGGCACACA CGGCTTTGTT GTGGGTCATA TCGTCCCTGA                          AGCAGTTGAA                                                                        7801   GGTGGGTTGA TTGGTTTAGT TGAAGATGAT GATATTATCG                          AAATTGATGC                                                                        7851   GGTGAATAAT AGTATTAGTT TAAAAGTTTC TAATGAAGAA                          ATTGCTAAAC                                                                        7901   GACGTGCCAA TTATCAAAAA CCAACCCCTA AAGCAACGCG                          TGGTGTTCTT                                                                        7951   GCAAAATTTG CCAAACTTAC GCGCCCCGCT AGTGAAGGTT                          GCGTTACAGA                                                                        8001   TTTATAGAAA GGTTTGAATG AAAAAAATAA AGTTAGAAAA                          ACCTACTTCC                                                                        8051   GGTTCCCAAC TTGTTCTCCA AACCTTAAAA GAACTTGGAG                          TAGAAATTAT                                                                        8101   TTTTGGTTAT CCTGGTGGGG CCATGCTCCC CTTGTATGAT                          GCGATTCATA                                                                        8151   ATTTTGAAGG AATTCAACAT ATTTTAGCCC GTCATGAGCA                          AGGAGCAACG                                                                        8201   CATGAAGCCG AAGGTTACGC TAAATCGTCT GGTAAAGTTG                          GTGTCGTCGT                                                                        8251   TGTTACGTCA GGACCAGGAG CGACTAATGC AGTAACCGGA                          ATTGCTGACG                                                                        8301   CTTATCTTGA TTCAGTCCCA TTGTTAGTTT TCACAGGTCA                          AGTTGGCCGT                                                                        8351   CAGTCAATTG GTAAAGATGC TTTTCAAGAA GCAGATACTG                          TTGGAATTAC                                                                        8401   AGCCCCAATT ACAAAATATA ATTATCAAAT TAGGGAAACC                          GCAGATATTC                                                                        8451   CAAGAATTGT TACAGAAGCC TATTATTTGG CAAGGACAGG                          ACGTCCTGGA                                                                        8501   CCAGTAGAAA TTGATTTACC AAAAGATGTT TCCACCCTTG                          AAGTCACTGA                                                                        8551   AATTAATGAC CCAAGCTTGA ATCTTCCTCA TTATCACGAA                          AGTGAAAAAG                                                                        8601   CGACTGATGA ACAATTGCAA GAATTACTGA CAGAACTTTC                          TGTCAGTAAA                                                                        8651   AAACCAGTCA TTATTGCTGG CGGAGGAATT AATTATTCTG                          GCTCAGTTGA                                                                        8701   TATTTTCAGA GCATTTGTCG AAAAATATCA AATTCCAGTT                          GTTTCTACAT                                                                        8751   TGCTTGGCTT AGGAACATTA CCAATCAGCC ACGAATTGCA                          ACTAGGAATG                                                                        8801   GCAGGAATGC ACGGTTCATA CGCTGCAAAT ATGGCTTTAG                          TTGAAGCTGA                                                                        8851   CTATATTATT AATTTGGGAT CACGTTTTGA CGATAGAGTT                          GTATCCAATC                                                                        8901   CTGCAAAATT TGCTAAAAAT GCTGTCGTTG CTCATATTGA                          TATTGACGCT                                                                        8951   GCTGAACTTG GCAAAATTGT AAAAACCGAT ATTCCAATCC                          TTTCTGATTT                                                                        9001   GAAAGCGGCT TTAAGCAGAC TTTTGCAATT AAATAAGGTC                          AGGACTGACT                                                                        9051   TTAATGATTG GATTAAAACT GTCATTGAAA ATAAAGAGAA                          AGCACCATTT                                                                        9101   ACTTATGAGC CCCAAAACCA TGATATCCGT CCACAGGAAA                          CAATTAAATT                                                                        9151   AATTGGAGAA TACACTCAAG GAGATGCAAT CATTGTAACT                          GACGTTGGGC                                                                        9201   AACATCAAAT GTGGGTGGCG CAATATTATC CTTATAAAAA                          TGCAAGGCAA                                                                        9251   CTTATTACTT CTGGGGGAAT GGGAACGATG GGCTTTGGCA                          TTCCTGCAGC                                                                        9301   AATCGGTGCA AAGCTGGCAC AGCCAAATAA AAATGTCATT                          GTTTTTGTTG                                                                        9351   GCGATGGTGG CTTTCAAATG ACTAATCAAG AATTAGCATT                          ACTTAATGGC                                                                        9401   TACGGTATTG CAATCAAAGT TGTGCTGATT AATAATCATT                          CATTGGGAAT                                                                        9451   GGTACGTCAA TGGCAAGAAT CATTCTATGA AGAGCGACGT                          TCACAATCGG                                                                        9501   TTTTTGATGT TGAACCCAAT TTTCAATTGT TAGCCGAAGC                          TTATGGCATC                                                                        9551   AAACATGTTA AGTTAGATAA TCCAAAAACT TTGGCTGATG                          ATTTAAAAAT                                                                        9601   TATTACAGAA GATGAGCCAA TGCTTATTGA AGTTCTAATT                          TCAAAATCTG                                                                        9651   AGCATGTTTT ACCAATGATA CCAGCTGGAT TACACAATGA                          CGAAATGATT                                                                        9701   GGACTTCATT TTACTGATAA GAATGAGGAG ATAGATAATG                          CGTAGAATGA                                                                        9751   TTATCGCAAA ACTTCATAAC GTGACAGGAA TTATGAATCG                          ATTTACCGCC                                                                        9801   GTTCTCAATC GAAGGCAAGT GAACATTCTC TCAATTACCG                          CTGGAGTTAC                                                                        9851   AGAAAGTCAA GACTTAACTC ATACCACTTT TGTTATTGAA                          GTTGATCATC                                                                        9901   TTGATGAAGT AGAACAAATC ATTAAACAAT TAAATCGCTT                          AATAGATGTA                                                                        9951   ATTGAAGTAG CTGATATTAC TGATTTTCCT CATGTAGAAC                          GTGAAGTCGT                                                                       10001   CTTGATTAAA GTATCAGCTC CACCGACCAT TAGGGCAGAA                          ATTTTTACAA                                                                       10051   TGATTGAACC TTTTAGAGTA AATGTAGTTG ATGTCAATCT                          GGAAAATGTC                                                                       10101   ACCATTCAAT TAACGGGTGA TTCAGCAAAA ATCGAAGCAC                          TTATTGAGGT                                                                       10151   TGTTAGTCCT TATGGCATTC TAAATATGGC TCGGACAGGT                          AGTGCAGGTT                                                                       10201   TTGAGCGTGG CTAAATTTAA ATAAGTTAAC AAATAAATAG                          AAAAATAGAG                                                                       10251   GAAACAAAAA TGGCAGTTAC AATGTATTAT GAAGATGATG                          TAGAAGTATC                                                                       10301   AGCACTTGCT GGAAAGCAAA TTGCAGTAAT CGGTTATGGT                          TCACAAGGAC                                                                       10351   ATGCTCACGC ACAGAATTTG CGTGATTCTG GTCACAACGT                          TATCATTGGT                                                                       10401   GTGCGCCACG GAAAATCTTT TGATAAAGCA AAAGAAGATG                          GCTTTGAAAC                                                                       10451   ATTTGAAGTA GGAGAAGCAG TAGCTAAAGC TGATGTTATT                          ATGGTTTTGG                                                                       10501   CACCAGATGA ACTTCAACAA TCCATTTATG AAGAGGACAT                          CAAACCAAAC                                                                       10551   TTGAAAGCAG GTTCAGCACT TGGTTTTGCT CACGGATTTA                          ATATCCATTT                                                                       10601   TGGCTATATT AAAGTACCAG AAGACGTTGA CGTCTTTATG                          GTTGCGCCTA                                                                       10651   AGGCTCCAGG TCACCTTGTC CGTCGGACTT ATACTGAAGG                          TTTTGGTACA                                                                       10701   CCAGCTTTGT TTGTTTCACA CCAAAATGCA AGTGGTCATG                          CGCGTGAAAT                                                                       10751   CGCAATGGAT TGGGCCAAAG GAATTGGTTG TGCTCGAGTG                          GGAATTATTG                                                                       10801   AAACAACTTT TAAAGAAGAA ACAGAAGAAG ATTTGTTTGG                          AGAACAAGCT                                                                       10851   GTTCTATGTG GAGGTTTGAC AGCACTTGTT GAAGCCGGTT                          TTGAAACACT                                                                       10901   GACAGAAGCT GGATACGCTG GCGAATTGGC TTACTTTGAA                          GTTTTGCACG                                                                       10951   AAATGAAATT GATTGTTGAC CTCATGTATG AAGGTGGTTT                          TACTAAAATG                                                                       11001   CGTCAATCCA TCTCAAATAC TGCTGAGTTT GGCGATTATG                          TGACTGGTCC                                                                       11051   ACGGATTATT ACTGACGAAG TTAAAAAGAA TATGAAGCTT                          GTTTTGGCTG                                                                       11101   ATATTCAATC TGGAAAATTT GCTCAAGATT TCGTTGATGA                          CTTCAAAGCG                                                                       11151   GGGCGTCCAA AATTAATAGC CTATCGCGAA GCTGCAAAAA                          ATCTTGAAAT                                                                       11201   TGAAAAAATT GGGGCAGAGC ACGTCAAGCA ATGCCATTCA                          CACAATCTGG                                                                       11251   TGATGACGAT GCCTTTAAAA TCTATCAGTA ATTTCTCTTA                          TTGATTGAAC                                                                       11301   AAAAACATAA AAGCATTTTA TGGAGGAATG ACATAAATGA                          TAAGTGCCAA                                                                       11351   AGAGGTTGAA GATGCCTATG ATTTGTTAAA AGCAGTTGTC                          ACTAAAACAC                                                                       11401   CTTTACAATT AGACCCTTAC CTTTCCAATA AATATCAAGC                          AAATATTTAC                                                                       11451   TTAAAAGAAG TTGTCACTAA AACACCTTTA CAATTAGACC                          CTTACCTTTC                                                                       11501   CAATAAATAT CAAGCAAATA TTTACTTAAA AGAAGAAAAC                          TTACAGAAAG                                                                       11551   TTCGTTCTTT TAAATTACGA GGAGCTTATT ATTCTATCAG                          TAAATTATCT                                                                       11601   GATGAGCAAC GCTCTAAAGG AGTGGTTTGT GCCTCAGCAG                          GAAATCATGC                                                                       11651   ACAAGGGGTT GCTTTTGCTG CAAATCAATT AAATATTTCT                          GCGACAATTT                                                                       11701   TTATGCCCGT TACCACACCT AACCAAAAAA TTTCACAAGT                          TAAATTTTTT                                                                       11751   GGCGAAAGTC ACGTAACAAT TCGTTTAATT GGTGATACTT                          TTGATGAATC                                                                       11801   AGCCAGAGCA GCAAAAGCTT TTTCTCAAGA TAATGACAAA                          CCATTTATAG                                                                       11851   ACCCTTTTGA TGATGAAAAT GTAATTGCTG GTCAAGGGAC                          AGTGGCTTTA                                                                       11901   GAAATTTTTG CGCAAGCTAA AAAACAAGGA ATAAGTTTAG                          ATAAGATTTT                                                                       11951   TGTACAGATT GGTGGAGGTG GTTTAATTGC AGGAATTACT                          GCCTACAGTA                                                                       12001   AGGAGCGCTA TCCCCAAACT GAAATTATCG GAGTTGAAGC                          AAAAGGGGCA                                                                       12051   ACAAGTATGA AAGCTGCCTA CTCTGCTGGT CAGCCCGTCA                          CCTTGGAACA                                                                       12101   CATTGATAAA TTTGCTGACG GAATTGCGGT TGCGACTGTC                          GGTCAGAAAA                                                                       12151   CTTACCAACT TATTAATGAC AAAGTGAAAC AATTGCTTGC                          GGTTGATGAA                                                                       12201   GGTTTAATTT CTCAAACCAT ACTCGAATTG TATTCAAAAT                          TAGGAATTGT                                                                       12251   CGCCGAGCCA GCAGGTGCAA CATCTGTTGC CGCACTTGAA                          CTTATTAAAG                                                                       12301   ATGAAATCAA GGGTAAAAAT ATCGTCTGTA TCATCAGCGG                          CGGAAATAAT                                                                       12351   GATATTAGTC GAATGCAAGA AATTGAAGAA AGAGCTTTGG                          TTTATGAAGG                                                                       12401   TCTAAAACAT TATTTTGTCA TTAACTTTCC TCAAAGACCA                          GGATCCTTAC                                                                       12451   GAACTTTTGT CAGTGATATT TTAGGGCCAA ATGATGATAT                          CACCCGATTT                                                                       12501   GAGTACATCA AAAGGGCTGA TAAAGGTAAA GGACCTTGTC                          TTGTTGGGAT                                                                       12551   TTTACTTTCA GATGCTAGTG ATTATGATTC ATTGATTAAT                          CGGATTGAAA                                                                       12601   GATTTGATAA TCGTTATGTT AACTTACGTG GAAATGATAG                          TTTATACGAA                                                                       12651   CTTTTGGTCT AACTAACCAA TTGGTTTGAG CCATTTTCTA                          GTTTCAATTC                                                                       12701   TCTTTAAATC ACTAGAAATT                                                ilvb   Length: 1728   September 22, 1993   11:08   Type: N                     Check: 9885 . .                                                                    1     ATGAAAAAAA TAAAGTTAGA AAAACCTACT TCCGGTTCCC                          AACTTGTTCT                                                                         51    CCAAACCTTA AAAGAACTTG GAGTAGAAAT TATTTTTGGT                          TATCCTGGTG                                                                        101    GGGCCATGCT CCCCTTGTAT GATGCGATTC ATAATTTTGA                          AGGAATTCAA                                                                        151    CATATTTTAG CCCGTCATGA GCAAGGAGCA ACGCATGAAG                          CCGAAGGTTA                                                                        201    CGCTAAATCG TCTGGTAAAG TTGGTGTCGT CGTTGTTACG                          TCAGGACCAG                                                                        251    GAGCGACTAA TGCAGTAACC GGAATTGCTG ACGCTTATCT                          TGATTCAGTC                                                                        301    CCATTGTTAG TTTTCACAGG TCAAGTTGGC CGTCAGTCAA                          TTGGTAAAGA                                                                        351    TGCTTTTCAA GAAGCAGATA CTGTTGGAAT TACAGCCCCA                          ATTACAAAAT                                                                        401    ATAATTATCA AATTAGGGAA ACCGCAGATA TTCCAAGAAT                          TGTTACAGAA                                                                        451    GCCTATTATT TGGCAAGGAC AGGACGTCCT GGACCAGTAG                          AAATTGATTT                                                                        501    ACCAAAAGAT GTTTCCACCC TTGAAGTCAC TGAAATTAAT                          GACCCAAGCT                                                                        551    TGAATCTTCC TCATTATCAC GAAAGTGAAA AAGCGACTGA                          TGAACAATTG                                                                        601    CAAGAATTAC TGACAGAACT TTCTGTCAGT AAAAAACCAG                          TCATTATTGC                                                                        651    TGGCGGAGGA ATTAATTATT CTGGCTCAGT TGATATTTTC                          AGAGCATTTG                                                                        701    TCGAAAAATA TCAAATTCCA GTTGTTTCTA CATTGCTTGG                          CTTAGGAACA                                                                        751    TTACCAATCA GCCACGAATT GCAACTAGGA ATGGCAGGAA                          TGCACGGTTC                                                                        801    ATACGCTGCA AATATGGCTT TAGTTGAAGC TGACTATATT                          ATTAATTTGG                                                                        851    GATCACGTTT TGACGATAGA GTTGTATCCA ATCCTGCAAA                          ATTTGCTAAA                                                                        901    AATGCTGTCG TTGCTCATAT TGATATTGAC GCTGCTGAAC                          TTGGCAAAAT                                                                        951    TGTAAAAACC GATATTCCAA TCCTTTCTGA TTTGAAAGCG                          GCTTTAAGCA                                                                        1001   GACTTTTGCA ATTAAATAAG GTCAGGACTG ACTTTAATGA                          TTGGATTAAA                                                                        1051   ACTGTCATTG AAAATAAAGA GAAAGCACCA TTTACTTATG                          AGCCCCAAAA                                                                        1101   CCATGATATC CGTCCACAGG AAACAATTAA ATTAATTGGA                          GAATACACTC                                                                        1151   AAGGAGATGC AATCATTGTA ACTGACGTTG GGCAACATCA                          AATGTGGGTG                                                                        1201   GCGCAATATT ATCCTTATAA AAATGCAAGG CAACTTATTA                          CTTCTGGGGG                                                                        1251   AATGGGAACG ATGGGCTTTG GCATTCCTGC AGCAATCGGT                          GCAAAGCTGG                                                                        1301   CACAGCCAAA TAAAAATGTC ATTGTTTTTG TTGGCGATGG                          TGGCTTTCAA                                                                        1351   ATGACTAATC AAGAATTAGC ATTACTTAAT GGCTACGGTA                          TTGCAATCAA                                                                        1401   AGTTGTGCTG ATTAATAATC ATTCATTGGG AATGGTACGT                          CAATGGCAAG                                                                        1451   AATCATTCTA TGAAGAGCGA CGTTCACAAT CGGTTTTTGA                          TGTTGAACCC                                                                        1501   AATTTTCAAT TGTTAGCCGA AGCTTATGGC ATCAAACATG                          TTAAGTTAGA                                                                        1551   TAATCCAAAA ACTTTGGCTG ATGATTTAAA AATTATTACA                          GAAGATGAGC                                                                        1601   CAATGCTTAT TGAAGTTCTA ATTTCAAAAT CTGAGCATGT                          TTTACCAATG                                                                        1651   ATACCAGCTG GATTACACAA TGACGAAATG ATTGGACTTC                          ATTTTACTGA                                                                        1701   TAAGAATGAG GAGATAGATA ATGCGTAG                                       ilvn   Length: 477   September 22, 1993   11:09   Type: N                      Check: 6061 . .                                                                    1     ATGCGTAGAA TGATTATCGC AAAACTTCAT AACGTGACAG                          GAATTATGAA                                                                         51    TCGATTTACC GCCGTTCTCA ATCGAAGGCA AGTGAACATT                          CTCTCAATTA                                                                        101    CCGCTGGAGT TACAGAAAGT CAAGACTTAA CTCATACCAC                          TTTTGTTATT                                                                        151    GAAGTTGATC ATCTTGATGA AGTAGAACAA ATCATTAAAC                          AATTAAATCG                                                                        201    CTTAATAGAT GTAATTGAAG TAGCTGATAT TACTGATTTT                          CCTCATGTAG                                                                        251    AACGTGAAGT CGTCTTGATT AAAGTATCAG CTCCACCGAC                          CATTAGGGCA                                                                        301    GAAATTTTTA CAATGATTGA ACCTTTTAGA GTAAATGTAG                          TTGATGTCAA                                                                        351    TCTGGAAAAT GTCACCATTC AATTAACGGG TGATTCAGCA                          AAAATCGAAG                                                                        401    CACTTATTGA GGTTGTTAGT CCTTATGGCA TTCTAAATAT                          GGCTCGGACA                                                                        451    GGTAGTGCAG GTTTTGAGCG TGGCTAA                                        ilvbn   Length: 2231   September 22, 1993   11:12   Type: N                    Check: 7704 . .                                                                    1     ATTTATAGAA AGGTTTGAAT GAAAAAAATA AAGTTAGAAA                          AACCTACTTC                                                                         51    CGGTTCCCAA CTTGTTCTCC AAACCTTAAA AGAACTTGGA                          GTAGAAATTA                                                                        101    TTTTTGGTTA TCCTGGTGGG GCCATGCTCC CCTTGTATGA                          TGCGATTCAT                                                                        151    AATTTTGAAG GAATTCAACA TATTTTAGCC CGTCATGAGC                          AAGGAGCAAC                                                                        201    GCATGAAGCC GAAGGTTACG CTAAATCGTC TGGTAAAGTT                          GGTGTCGTCG                                                                        251    TTGTTACGTC AGGACCAGGA GCGACTAATG CAGTAACCGG                          AATTGCTGAC                                                                        301    GCTTATCTTG ATTCAGTCCC ATTGTTAGTT TTCACAGGTC                          AAGTTGGCCG                                                                        351    TCAGTCAATT GGTAAAGATG CTTTTCAAGA AGCAGATACT                          GTTGGAATTA                                                                        401    CAGCCCCAAT TACAAAATAT AATTATCAAA TTAGGGAAAC                          CGCAGATATT                                                                        451    CCAAGAATTG TTACAGAAGC CTATTATTTG GCAAGGACAG                          GACGTCCTGG                                                                        501    ACCAGTAGAA ATTGATTTAC CAAAAGATGT TTCCACCCTT                          GAAGTCACTG                                                                        551    AAATTAATGA CCCAAGCTTG AATCTTCCTC ATTATCACGA                          AAGTGAAAAA                                                                        601    GCGACTGATG AACAATTGCA AGAATTACTG ACAGAACTTT                          CTGTCAGTAA                                                                        651    AAAACCAGTC ATTATTGCTG GCGGAGGAAT TAATTATTCT                          GGCTCAGTTG                                                                        701    ATATTTTCAG AGCATTTGTC GAAAAATATC AAATTCCAGT                          TGTTTCTACA                                                                        751    TTGCTTGGCT TAGGAACATT ACCAATCAGC CACGAATTGC                          AACTAGGAAT                                                                        801    GGCAGGAATG CACGGTTCAT ACGCTGCAAA TATGGCTTTA                          GTTGAAGCTG                                                                        851    ACTATATTAT TAATTTGGGA TCACGTTTTG ACGATAGAGT                          TGTATCCAAT                                                                        901    CCTGCAAAAT TTGCTAAAAA TGCTGTCGTT GCTCATATTG                          ATATTGACGC                                                                        951    TGCTGAACTT GGCAAAATTG TAAAAACCGA TATTCCAATC                          CTTTCTGATT                                                                       1001    TGAAAGCGGC TTTAAGCAGA CTTTTGCAAT TAAATAAGGT                          CAGGACTGAC                                                                       1051    TTTAATGATT GGATTAAAAC TGTCATTGAA AATAAAGAGA                          AAGCACCATT                                                                       1101    TACTTATGAG CCCCAAAACC ATGATATCCG TCCACAGGAA                          ACAATTAAAT                                                                       1151    TAATTGGAGA ATACACTCAA GGAGATGCAA TCATTGTAAC                          TGACGTTGGG                                                                       1201    CAACATCAAA TGTGGGTGGC GCAATATTAT CCTTATAAAA                          ATGCAAGGCA                                                                       1251    ACTTATTACT TCTGGGGGAA TGGGAACGAT GGGCTTTGGC                          ATTCCTGCAG                                                                       1301    CAATCGGTGC AAAGCTGGCA CAGCCAAATA AAAATGTCAT                          TGTTTTTGTT                                                                       1351    GGCGATGGTG GCTTTCAAAT GACTAATCAA GAATTAGCAT                          TACTTAATGG                                                                       1401    CTACGGTATT GCAATCAAAG TTGTGCTGAT TAATAATCAT                          TCATTGGGAA                                                                       1451    TGGTACGTCA ATGGCAAGAA TCATTCTATG AAGAGCGACG                          TTCACAATCG                                                                       1501    GTTTTTGATG TTGAACCCAA TTTTCAATTG TTAGCCGAAG                          CTTATGGCAT                                                                       1551    CAAACATGTT AAGTTAGATA ATCCAAAAAC TTTGGCTGAT                          GATTTAAAAA                                                                       1601    TTATTACAGA AGATGAGCCA ATGCTTATTG AAGTTCTAAT                          TTCAAAATCT                                                                       1651    GAGCATGTTT TACCAATGAT ACCAGCTGGA TTACACAATG                          ACGAAATGAT                                                                       1701    TGGACTTCAT TTTACTGATA AGAATGAGGA GATAGATAAT                          GCGTAGAATG                                                                       1751    ATTATCGCAA AACTTCATAA CGTGACAGGA ATTATGAATC                          GATTTACCGC                                                                       1801    CGTTCTCAAT CGAAGGCAAG TGAACATTCT CTCAATTACC                          GCTGGAGTTA                                                                       1851    CAGAAAGTCA AGACTTAACT CATACCACTT TTGTTATTGA                          AGTTGATCAT                                                                       1901    CTTGATGAAG TAGAACAAAT CATTAAACAA TTAAATCGCT                          TAATAGATGT                                                                       1951    AATTGAAGTA GCTGATATTA CTGATTTTCC TCATGTAGAA                          CGTGAAGTCG                                                                       2001    TCTTGATTAA AGTATCAGCT CCACCGACCA TTAGGGCAGA                          AATTTTTACA                                                                       2051    ATGATTGAAC CTTTTAGAGT AAATGTAGTT GATGTCAATC                          TGGAAAATGT                                                                       2101    CACCATTCAA TTAACGGGTG ATTCAGCAAA AATCGAAGCA                          CTTATTGAGG                                                                       2151    TTGTTAGTCC TTATGGCATT CTAAATATGG CTCGGACAGG                          TAGTGCAGGT                                                                       2201    TTTGAGCGTG GCTAAATTTA AATAAGTTAA C                                   __________________________________________________________________________

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 16                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 513 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: LEUA                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       MetArgLysIleGluPhePheAspThrSerLeuArgAspGlyGluGln                               151015                                                                         ThrProGlyValSerPheSerIleSerGluLysValThrIleAlaLys                               202530                                                                         GlnLeuGluLysTrpArgIleSerValIleGluAlaGlyPheSerAla                               354045                                                                         AlaSerProAspSerPheGluAlaValLysGlnIleAlaAspSerLeu                               505560                                                                         AsnAspThrAlaValThrAlaLeuAlaArgCysValIleSerAspIle                               65707580                                                                       AspLysAlaValGluAlaValLysGlyAlaLysTyrProGlnIleHis                               859095                                                                         ValPheIleAlaThrSerProIleHisMetLysTyrLysLeuLysIle                               100105110                                                                      SerProGluGluValLeuLysAsnIleAspLysCysValArgTyrAla                               115120125                                                                      ArgGluArgValGluValValGluPheSerProGluAspAlaThrArg                               130135140                                                                      ThrGluLeuAsnPheLeuLeuGluAlaValGlnThrAlaValAspAla                               145150155160                                                                   GlyAlaThrTyrIleAsnIleProAspThrValGlyTyrThrThrPro                               165170175                                                                      GluGluTyrGlyLysIlePheLysPheLeuIleAspAsnThrLysSer                               180185190                                                                      AspArgGluIleIlePheSerProHisCysHisAspAspLeuGlyMet                               195200205                                                                      AlaValAlaAsnSerLeuAlaAlaIleLysAlaGlyAlaGlyArgVal                               210215220                                                                      GluGlyThrValAsnGlyIleGlyGluArgAlaGlyAsnAlaAlaLeu                               225230235240                                                                   GluGluIleAlaValAlaLeuHisIleArgLysAspPheTyrGlnAla                               245250255                                                                      GlnSerProLeuLysLeuSerGluThrAlaAlaThrAlaGluLeuIle                               260265270                                                                      SerGlnPheSerGlyIleAlaIleProLysAsnLysAlaIleValGly                               275280285                                                                      AlaAsnAlaPheAlaHisGluSerGlyIleHisGlnAspGlyValLeu                               290295300                                                                      LysAsnAlaGluThrTyrGluIleIleThrProGluLeuValGlyIle                               305310315320                                                                   LysHisAsnSerLeuProLeuGlyLysLeuSerGlyArgHisAlaPhe                               325330335                                                                      SerGluLysLeuThrGluLeuAsnIleAlaTyrAspAspGluSerLeu                               340345350                                                                      AlaIleLeuPheGluLysPheLysLysLeuAlaAspLysLysLysGlu                               355360365                                                                      IleThrAspAlaAspIleHisAlaLeuPheThrGlyGluThrValLys                               370375380                                                                      AsnLeuAlaGlyPheIleLeuAspAsnValGlnIleAspGlyHisLys                               385390395400                                                                   AlaLeuValGlnLeuLysAsnGlnGluGluGluIleTyrValSerGln                               405410415                                                                      GlyGluGlySerGlySerValAspAlaIlePheLysAlaIleAspLys                               420425430                                                                      ValPheAsnHisGlnLeuLysLeuIleSerTyrSerValAspAlaVal                               435440445                                                                      ThrAspGlyIleAspAlaGlnAlaThrThrLeuValSerValGluAsn                               450455460                                                                      LeuSerThrGlyThrIlePheAsnAlaLysGlyValAspTyrAspVal                               465470475480                                                                   LeuLysGlySerAlaIleAlaTyrMetAsnAlaAsnValLeuValGln                               485490495                                                                      LysGluAsnLeuGlnGlyLysValGluGlnIleSerAlaHisAspGly                               500505510                                                                      Ile                                                                            (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 345 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: LEUB                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       LeuSerLysLysIleValThrLeuAlaGlyAspGlyIleGlyProGlu                               151015                                                                         IleMetSerAlaGlyLeuSerValLeuLysAlaValSerLysLysIle                               202530                                                                         AspPheGluTyrGluLeuGluAlaLysAspPheGlyGlyIleAlaIle                               354045                                                                         AspLysHisGlyHisProLeuProGluGluThrLeuGlnAlaValLys                               505560                                                                         AsnAlaAspAlaIleLeuLeuAlaAlaIleGlyHisProLysTyrAsn                               65707580                                                                       AsnAlaLysValArgProGluGlnGlyLeuLeuAlaLeuArgLysGlu                               859095                                                                         LeuGlyLeuTyrAlaAsnValArgProLeuLysIleTyrProAlaLeu                               100105110                                                                      LysLysLeuSerProIleArgAsnValGluAsnValAspPheLeuVal                               115120125                                                                      IleArgGluLeuThrGlyGlyIleTyrPheGlyGlnHisGluLeuAla                               130135140                                                                      AspAspLysAlaArgAspValAsnAspTyrSerAlaAspGluIleArg                               145150155160                                                                   ArgIleLeuHisPheAlaPheLysSerAlaGlnSerArgProArgLys                               165170175                                                                      LeuLeuThrSerValAspLysGlnAsnValLeuAlaThrSerLysLeu                               180185190                                                                      TrpArgLysMetAlaAspGluIleAlaAspGluTyrProAspValArg                               195200205                                                                      LeuGluHisGlnLeuValAspSerCysAlaMetLeuLeuIleThrAsn                               210215220                                                                      ProGlnGlnPheAspValIleValThrGluAsnLeuPheGlyAspIle                               225230235240                                                                   LeuSerAspGluAlaSerSerLeuAlaGlySerLeuGlyValMetPro                               245250255                                                                      SerSerSerHisGlyPheAsnGlyLeuAlaLeuTyrGluProIleHis                               260265270                                                                      GlySerAlaProAspIleAlaGlyLysGlyIleAlaAsnProValSer                               275280285                                                                      MetIleLeuSerIleAlaMetMetLeuArgGluSerPheGlyGlnGlu                               290295300                                                                      AspGlyAlaAlaMetIleGluLysAlaValThrGlnThrPheThrAsp                               305310315320                                                                   GlyIleLeuThrLysAspLeuGlyGlyThrAlaThrThrLysGluMet                               325330335                                                                      ThrGluAlaIleLeuLysAsnCysGln                                                    340345                                                                         (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 460 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: LEUC                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       MetSerGlyLysThrIlePheAspLysLeuTrpAspGlnHisValIle                               151015                                                                         AlaGlyAsnGluGlyGluProGlnLeuLeuTyrIleAspLeuHisVal                               202530                                                                         IleHisGluValThrSerProGlnAlaPheGlnGlyLeuArgGluAla                               354045                                                                         GlyArgArgValArgArgLysAspLeuThrTyrGlyThrLeuAspHis                               505560                                                                         AsnValProThrGlnAsnIlePheAsnIleGlnAspLeuIleSerLys                               65707580                                                                       LysGlnIleAspThrPheThrLysAsnValLysGluPheAspValPro                               859095                                                                         AlaGluThrHisGlyGlyLysGlyGlnGlyIleValHisMetValAla                               100105110                                                                      ProGluSerGlyArgThrGlnProGlyLysThrIleValCysGlyAsp                               115120125                                                                      SerHisThrAlaThrAsnGlyAlaPheGlyAlaIleAlaPheGlyIle                               130135140                                                                      GlyThrSerGluValGluHisValLeuAlaThrGlnThrIleTrpGln                               145150155160                                                                   ValLysProLysArgMetLysIleGluPheGlnGlyHisProGlnLys                               165170175                                                                      GlyIleTyrSerLysAspPheIleLeuAlaLeuIleAlaLysTyrGly                               180185190                                                                      ValAspAlaGlyValGlyTyrAlaValGluTyrSerGlyAspAlaIle                               195200205                                                                      SerAspLeuSerMetGluGluArgMetThrIleCysAsnMetSerIle                               210215220                                                                      GluPheGlyAlaLysIleGlyLeuMetAsnProAspGluLysThrTyr                               225230235240                                                                   AspTyrValLysGlyArgGluHisAlaProLysAsnPheAspGluAla                               245250255                                                                      ValSerLysTrpGluLysLeuValSerAspSerAspAlaGlnTyrAsp                               260265270                                                                      LysIleLeuSerLeuAspValSerGlnLeuLysProMetValThrTrp                               275280285                                                                      GlyThrAsnProGlyMetGlyLeuGluPheGlyGluLysPheProGlu                               290295300                                                                      IleAsnAsnAspLeuAsnTyrGluArgAlaTyrGlnTyrMetAspLeu                               305310315320                                                                   LysProGlyGlnThrAlaSerAspIleAspLeuGlyTyrIlePheIle                               325330335                                                                      GlySerCysThrAsnAlaArgLeuGlyAspLeuGluGluAlaAlaLys                               340345350                                                                      IleIleGlyAspArgHisIleAlaAspGlyLeuThrGlyIleValVal                               355360365                                                                      ProGlySerArgProValLysGluAlaAlaGluAlaGlnGlyLeuAsp                               370375380                                                                      LysIlePheLysGluAlaGlyPheGluTrpArgGluProGlyCysSer                               385390395400                                                                   AlaCysLeuGlyMetAsnProAspGlnIleProGluTyrValHisCys                               405410415                                                                      AlaSerThrSerAsnArgAsnPheGluGlyArgGlnGlyHisAsnAla                               420425430                                                                      ArgThrHisLeuCysSerProAlaMetAlaAlaAlaAlaAlaIleAla                               435440445                                                                      GlyLysPheValAspValArgMetLeuValThrAsp                                           450455460                                                                      (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 191 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: LEUD                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       MetGluLysPheThrIleTyrLysGlyThrSerValProValMetAsn                               151015                                                                         AspAsnIleAspThrAspGlnIleIleProLysGlnPheLeuLysAla                               202530                                                                         IleAspLysLysGlyPheGlyLysAsnLeuPheTyrGluTrpArgTyr                               354045                                                                         LeuLysAspTyrAspGluAsnProAspPheIleLeuAsnAlaProLys                               505560                                                                         TyrLysLysAlaSerLeuLeuIleSerGlyAspAsnPheGlySerGly                               65707580                                                                       SerSerArgGluHisAlaAlaTrpAlaLeuSerAspTyrGlyPheArg                               859095                                                                         AlaIleIleAlaGlySerTyrSerAspIlePheTyrAsnAsnAlaLeu                               100105110                                                                      LysAsnGlyLeuLeuProIleLysGlnProArgGluValLeuAsnGln                               115120125                                                                      LeuThrLysLeuSerSerGlnGluGluIleThrIleAspLeuProHis                               130135140                                                                      GlnLeuIleIleThrSerLeuGlyAspPheHisPheGluIleAspPro                               145150155160                                                                   IleTrpLysAspLysLeuIleAsnGlyLeuAspAspIleGlyIleThr                               165170175                                                                      LeuGlnTyrGluGluAlaIleSerAlaTyrGluGlnLysAsnGln                                  180185190                                                                      (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 259 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ORF2                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       MetThrIleIleAsnLeuLysAsnValAsnLeuThrArgAsnLysLys                               151015                                                                         GluIleLeuLysAspIleThrTrpLysValAsnProGlyGluAsnTrp                               202530                                                                         ValIleLeuGlyLeuAsnGlySerGlyLysSerSerLeuLeuLysLeu                               354045                                                                         IleLeuAlaGluGluTrpLysThrSerGlyGluIleThrValLeuAsn                               505560                                                                         ThrGlnPheArgAsnGlyGluIleProLysLeuArgLysArgIleSer                               65707580                                                                       ValValGlySerPheIleAlaGluArgPheGlnProAsnIleLysAla                               859095                                                                         GluAsnLeuValTyrThrGlyLysPheAsnSerSerMetLeuTyrLys                               100105110                                                                      ProTyrThrAspGlnGluLeuAspGluAlaArgGlnLeuLeuArgGln                               115120125                                                                      MetGlyAlaLysSerLeuIleGlyArgAsnTyrAlaSerLeuSerGln                               130135140                                                                      GlyGluLysGlnValLeuLeuIleAlaArgSerLeuIleLeuLysPro                               145150155160                                                                   GluLeuLeuIleLeuAspGluAlaThrAsnGlyLeuAspLeuPheAla                               165170175                                                                      LysGluLysLeuLeuLysGlnLeuGlnGlnIleAsnGlnLeuLysThr                               180185190                                                                      AlaProThrLeuIleTyrIleSerHisHisProAspGluIleThrAsp                               195200205                                                                      IlePheThrHisLeuLeuLeuLeuArgGluGlyLysValIleGlnSer                               210215220                                                                      GlyLysLysGluAsnLeuLeuAsnGluLysIleLeuThrAspPheTyr                               225230235240                                                                   GlnGluLysValGluValHisArgPheGluGlnLysTyrPheValIle                               245250255                                                                      ProAlaAsn                                                                      (2) INFORMATION FOR SEQ ID NO: 6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 570 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ILVD                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       MetGluPheLysTyrAsnGlyLysValGluSerValGluLeuAsnLys                               151015                                                                         TyrSerLysThrLeuThrProArgSerThrGlnProAlaThrGlnAla                               202530                                                                         MetTyrTyrGlyIleGlyPheLysAspGluAspPheLysLysAlaGln                               354045                                                                         ValGlyIleValSerMetAspTrpAspGlyAsnProCysAsnMetHis                               505560                                                                         LeuGlyThrLeuGlySerLysIleLysSerSerValAsnGlnThrAsp                               65707580                                                                       GlyLeuIleGlyLeuGlnPheHisThrIleGlyValSerAspGlyIle                               859095                                                                         AlaAsnGlyLysLeuGlyMetArgTyrSerLeuValSerArgGluVal                               100105110                                                                      IleAlaAspSerIleGluThrAsnAlaGlyAlaGluTyrTyrAspAla                               115120125                                                                      IleValAlaIleProGlyCysAspLysAsnMetProGlySerIleIle                               130135140                                                                      GlyMetAlaArgLeuAsnArgProSerIleMetValTyrGlyGlyThr                               145150155160                                                                   IleGluHisGlyGluTyrLysGlyGluLysLeuAsnIleValSerAla                               165170175                                                                      PheGluAlaLeuGlyGlnLysIleThrGlyAsnIleSerAspGluAsp                               180185190                                                                      TyrHisGlyValIleCysAsnAlaIleProGlyGlnGlyAlaCysGly                               195200205                                                                      GlyMetTyrThrAlaAsnThrLeuAlaAlaAlaIleGluThrLeuGly                               210215220                                                                      MetSerLeuProTyrSerSerSerAsnProAlaValSerGlnGluLys                               225230235240                                                                   GlnGluGluCysAspAspIleGlyLeuAlaIleLysAsnLeuLeuGlu                               245250255                                                                      LysAspIleLysProSerAspIleMetThrLysGluAlaPheGluAsn                               260265270                                                                      AlaIleThrIleValMetValLeuGlyGlySerThrAsnAlaValLeu                               275280285                                                                      HisIleIleAlaMetAlaAsnAlaIleGlyValGluIleThrGlnAsp                               290295300                                                                      AspPheGlnArgIleSerAspIleIleProValLeuGlyAspPheLys                               305310315320                                                                   ProSerGlyLysTyrMetMetGluAspLeuHisLysIleGlyGlyLeu                               325330335                                                                      ProAlaValLeuLysTyrLeuLeuLysGluGlyLysLeuHisGlyAsp                               340345350                                                                      CysLeuThrValThrGlyLysThrLeuAlaGluAsnValGluThrAla                               355360365                                                                      LeuAspLeuAspPheAspSerGlnAspIleMetArgProLeuLysAsn                               370375380                                                                      ProIleLysAlaThrGlyHisLeuGlnIleLeuTyrGlyAsnLeuAla                               385390395400                                                                   GlnGlyGlySerValAlaLysIleSerGlyLysGluGlyGluPhePhe                               405410415                                                                      LysGlyThrAlaArgValPheAspGlyGluGlnHisPheIleAspGly                               420425430                                                                      IleGluSerGlyArgLeuHisAlaGlyAspValAlaValIleArgAsn                               435440445                                                                      IleGlyProValGlyGlyProGlyMetProGluMetLeuLysProThr                               450455460                                                                      SerAlaLeuIleGlyAlaGlyLeuGlyLysSerCysAlaLeuIleThr                               465470475480                                                                   AspGlyArgPheSerGlyGlyThrHisGlyPheValValGlyHisIle                               485490495                                                                      ValProGluAlaValGluGlyGlyLeuIleGlyLeuValGluAspAsp                               500505510                                                                      AspIleIleGluIleAspAlaValAsnAsnSerIleSerLeuLysVal                               515520525                                                                      SerAsnGluGluIleAlaLysArgArgAlaAsnTyrGlnLysProThr                               530535540                                                                      ProLysAlaThrArgGlyValLeuAlaLysPheAlaLysLeuThrArg                               545550555560                                                                   ProAlaSerGluGlyCysValThrAspLeu                                                 565570                                                                         (2) INFORMATION FOR SEQ ID NO: 7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 575 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ILVB                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       MetLysLysIleLysLeuGluLysProThrSerGlySerGlnLeuVal                               151015                                                                         LeuGlnThrLeuLysGluLeuGlyValGluIleIlePheGlyTyrPro                               202530                                                                         GlyGlyAlaMetLeuProLeuTyrAspAlaIleHisAsnPheGluGly                               354045                                                                         IleGlnHisIleLeuAlaArgHisGluGlnGlyAlaThrHisGluAla                               505560                                                                         GluGlyTyrAlaLysSerSerGlyLysValGlyValValValValThr                               65707580                                                                       SerGlyProGlyAlaThrAsnAlaValThrGlyIleAlaAspAlaTyr                               859095                                                                         LeuAspSerValProLeuLeuValPheThrGlyGlnValGlyArgGln                               100105110                                                                      SerIleGlyLysAspAlaPheGlnGluAlaAspThrValGlyIleThr                               115120125                                                                      AlaProIleThrLysTyrAsnTyrGlnIleArgGluThrAlaAspIle                               130135140                                                                      ProArgIleValThrGluAlaTyrTyrLeuAlaArgThrGlyArgPro                               145150155160                                                                   GlyProValGluIleAspLeuProLysAspValSerThrLeuGluVal                               165170175                                                                      ThrGluIleAsnAspProSerLeuAsnLeuProHisTyrHisGluSer                               180185190                                                                      GluLysAlaThrAspGluGlnLeuGlnGluLeuLeuThrGluLeuSer                               195200205                                                                      ValSerLysLysProValIleIleAlaGlyGlyGlyIleAsnTyrSer                               210215220                                                                      GlySerValAspIlePheArgAlaPheValGluLysTyrGlnIlePro                               225230235240                                                                   ValValSerThrLeuLeuGlyLeuGlyThrLeuProIleSerHisGlu                               245250255                                                                      LeuGlnLeuGlyMetAlaGlyMetHisGlySerTyrAlaAlaAsnMet                               260265270                                                                      AlaLeuValGluAlaAspTyrIleIleAsnLeuGlySerArgPheAsp                               275280285                                                                      AspArgValValSerAsnProAlaLysPheAlaLysAsnAlaValVal                               290295300                                                                      AlaHisIleAspIleAspAlaAlaGluLeuGlyLysIleValLysThr                               305310315320                                                                   AspIleProIleLeuSerAspLeuLysAlaAlaLeuSerArgLeuLeu                               325330335                                                                      GlnLeuAsnLysValArgThrAspPheAsnAspTrpIleLysThrVal                               340345350                                                                      IleGluAsnLysGluLysAlaProPheThrTyrGluProGlnAsnHis                               355360365                                                                      AspIleArgProGlnGluThrIleLysLeuIleGlyGluTyrThrGln                               370375380                                                                      GlyAspAlaIleIleValThrAspValGlyGlnHisGlnMetTrpVal                               385390395400                                                                   AlaGlnTyrTyrProTyrLysAsnAlaArgGlnLeuIleThrSerGly                               405410415                                                                      GlyMetGlyThrMetGlyPheGlyIleProAlaAlaIleGlyAlaLys                               420425430                                                                      LeuAlaGlnProAsnLysAsnValIleValPheValGlyAspGlyGly                               435440445                                                                      PheGlnMetThrAsnGlnGluLeuAlaLeuLeuAsnGlyTyrGlyIle                               450455460                                                                      AlaIleLysValValLeuIleAsnAsnHisSerLeuGlyMetValArg                               465470475480                                                                   GlnTrpGlnGluSerPheTyrGluGluArgArgSerGlnSerValPhe                               485490495                                                                      AspValGluProAsnPheGlnLeuLeuAlaGluAlaTyrGlyIleLys                               500505510                                                                      HisValLysLeuAspAsnProLysThrLeuAlaAspAspLeuLysIle                               515520525                                                                      IleThrGluAspGluProMetLeuIleGluValLeuIleSerLysSer                               530535540                                                                      GluHisValLeuProMetIleProAlaGlyLeuHisAsnAspGluMet                               545550555560                                                                   IleGlyLeuHisPheThrAspLysAsnGluGluIleAspAsnAla                                  565570575                                                                      (2) INFORMATION FOR SEQ ID NO: 8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 158 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ILVN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       MetArgArgMetIleIleAlaLysLeuHisAsnValThrGlyIleMet                               151015                                                                         AsnArgPheThrAlaValLeuAsnArgArgGlnValAsnIleLeuSer                               202530                                                                         IleThrAlaGlyValThrGluSerGlnAspLeuThrHisThrThrPhe                               354045                                                                         ValIleGluValAspHisLeuAspGluValGluGlnIleIleLysGln                               505560                                                                         LeuAsnArgLeuIleAspValIleGluValAlaAspIleThrAspPhe                               65707580                                                                       ProHisValGluArgGluValValLeuIleLysValSerAlaProPro                               859095                                                                         ThrIleArgAlaGluIlePheThrMetIleGluProPheArgValAsn                               100105110                                                                      ValValAspValAsnLeuGluAsnValThrIleGlnLeuThrGlyAsp                               115120125                                                                      SerAlaLysIleGluAlaLeuIleGluValValSerProTyrGlyIle                               130135140                                                                      LeuAsnMetAlaArgThrGlySerAlaGlyPheGluArgGly                                     145150155                                                                      (2) INFORMATION FOR SEQ ID NO: 9:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 344 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ILVC                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                       MetAlaValThrMetTyrTyrGluAspAspValGluValSerAlaLeu                               151015                                                                         AlaGlyLysGlnIleAlaValIleGlyTyrGlySerGlnGlyHisAla                               202530                                                                         HisAlaGlnAsnLeuArgAspSerGlyHisAsnValIleIleGlyVal                               354045                                                                         ArgHisGlyLysSerPheAspLysAlaLysGluAspGlyPheGluThr                               505560                                                                         PheGluValGlyGluAlaValAlaLysAlaAspValIleMetValLeu                               65707580                                                                       AlaProAspGluLeuGlnGlnSerIleTyrGluGluAspIleLysPro                               859095                                                                         AsnLeuLysAlaGlySerAlaLeuGlyPheAlaHisGlyPheAsnIle                               100105110                                                                      HisPheGlyTyrIleLysValProGluAspValAspValPheMetVal                               115120125                                                                      AlaProLysAlaProGlyHisLeuValArgArgThrTyrThrGluGly                               130135140                                                                      PheGlyThrProAlaLeuPheValSerHisGlnAsnAlaSerGlyHis                               145150155160                                                                   AlaArgGluIleAlaMetAspTrpAlaLysGlyIleGlyCysAlaArg                               165170175                                                                      ValGlyIleIleGluThrThrPheLysGluGluThrGluGluAspLeu                               180185190                                                                      PheGlyGluGlnAlaValLeuCysGlyGlyLeuThrAlaLeuValGlu                               195200205                                                                      AlaGlyPheGluThrLeuThrGluAlaGlyTyrAlaGlyGluLeuAla                               210215220                                                                      TyrPheGluValLeuHisGluMetLysLeuIleValAspLeuMetTyr                               225230235240                                                                   GluGlyGlyPheThrLysMetArgGlnSerIleSerAsnThrAlaGlu                               245250255                                                                      PheGlyAspTyrValThrGlyProArgIleIleThrAspGluValLys                               260265270                                                                      LysAsnMetLysLeuValLeuAlaAspIleGlnSerGlyLysPheAla                               275280285                                                                      GlnAspPheValAspAspPheLysAlaGlyArgProLysLeuIleAla                               290295300                                                                      TyrArgGluAlaAlaLysAsnLeuGluIleGluLysIleGlyAlaGlu                               305310315320                                                                   HisValLysGlnCysHisSerHisAsnLeuValMetThrMetProLeu                               325330335                                                                      LysSerIleSerAsnPheSerTyr                                                       340                                                                            (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 441 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ILVA                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      MetIleSerAlaLysGluValGluAspAlaTyrAspLeuLeuLysAla                               151015                                                                         ValValThrLysThrProLeuGlnLeuAspProTyrLeuSerAsnLys                               202530                                                                         TyrGlnAlaAsnIleTyrLeuLysGluValValThrLysThrProLeu                               354045                                                                         GlnLeuAspProTyrLeuSerAsnLysTyrGlnAlaAsnIleTyrLeu                               505560                                                                         LysGluGluAsnLeuGlnLysValArgSerPheLysLeuArgGlyAla                               65707580                                                                       TyrTyrSerIleSerLysLeuSerAspGluGlnArgSerLysGlyVal                               859095                                                                         ValCysAlaSerAlaGlyAsnHisAlaGlnGlyValAlaPheAlaAla                               100105110                                                                      AsnGlnLeuAsnIleSerAlaThrIlePheMetProValThrThrPro                               115120125                                                                      AsnGlnLysIleSerGlnValLysPhePheGlyGluSerHisValThr                               130135140                                                                      IleArgLeuIleGlyAspThrPheAspGluSerAlaArgAlaAlaLys                               145150155160                                                                   AlaPheSerGlnAspAsnAspLysProPheIleAspProPheAspAsp                               165170175                                                                      GluAsnValIleAlaGlyGlnGlyThrValAlaLeuGluIlePheAla                               180185190                                                                      GlnAlaLysLysGlnGlyIleSerLeuAspLysIlePheValGlnIle                               195200205                                                                      GlyGlyGlyGlyLeuIleAlaGlyIleThrAlaTyrSerLysGluArg                               210215220                                                                      TyrProGlnThrGluIleIleGlyValGluAlaLysGlyAlaThrSer                               225230235240                                                                   MetLysAlaAlaTyrSerAlaGlyGlnProValThrLeuGluHisIle                               245250255                                                                      AspLysPheAlaAspGlyIleAlaValAlaThrValGlyGlnLysThr                               260265270                                                                      TyrGlnLeuIleAsnAspLysValLysGlnLeuLeuAlaValAspGlu                               275280285                                                                      GlyLeuIleSerGlnThrIleLeuGluLeuTyrSerLysLeuGlyIle                               290295300                                                                      ValAlaGluProAlaGlyAlaThrSerValAlaAlaLeuGluLeuIle                               305310315320                                                                   LysAspGluIleLysGlyLysAsnIleValCysIleIleSerGlyGly                               325330335                                                                      AsnAsnAspIleSerArgMetGlnGluIleGluGluArgAlaLeuVal                               340345350                                                                      TyrGluGlyLeuLysHisTyrPheValIleAsnPheProGlnArgPro                               355360365                                                                      GlySerLeuArgThrPheValSerAspIleLeuGlyProAsnAspAsp                               370375380                                                                      IleThrArgPheGluTyrIleLysArgAlaAspLysGlyLysGlyPro                               385390395400                                                                   CysLeuValGlyIleLeuLeuSerAspAlaSerAspTyrAspSerLeu                               405410415                                                                      IleAsnArgIleGluArgPheAspAsnArgTyrValAsnLeuArgGly                               420425430                                                                      AsnAspSerLeuTyrGluLeuLeuVal                                                    435440                                                                         (2) INFORMATION FOR SEQ ID NO: 11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12720 base pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                      TAAAACTCGATAATCTTGAGTCATAATTTCTCCTTAATCTTATTAGTACATTAGAATCCA60                 TTATAATTTAATCATTTTATGTCTACCTAAAGCAACAAAATTGCTTGTATATTTTCTAAC120                AAGCTTAATTATGTGGATTTAATTGAATATTAAAGGGAGAAGTTGTAATCTATTTGTTGT180                TAAATTCTTGTTAATACAAATAAATTTATTAAATATTATTATTTTATTGACAATTTAAAA240                TATTAAGAGTATTATAATGTAAATTAACAAAAAAAAGAGGAACTTGAAATGACATACACA300                CAATTTTCATTGTTGTTGATCAAGGTGGACCTACATTAGCTTTTTTGGCTAAAATATGTG360                GGTCCTGTTTGGCGATAGTCATTTCGAGGACCGAGAGACGTCCTCACGGGCGTCTTTTTT420                GTTTCTTAATAAAAAATAGAGGTAATATTATGCGAAAAATTGAATTCTTTGACACAAGTT480                TGAGAGATGGCGAACAGACACCGGGCGTTAGTTTCTCCATTTCAGAAAAAGTAACGATTG540                CTAAACAACTGGAAAAATGGAGGATTTCTGTCATAGAGGCTGGTTTTTCTGCGGCAAGTC600                CAGATAGTTTTGAAGCAGTAAAGCAAATTGCTGATTCTTTGAATGATACGGCTGTCACTG660                CATTAGCTCGCTGTGTTATTTCAGATATCGATAAAGCGGTTGAAGCGGTAAAGGGGGCTA720                AATATCCGCAAATTCATGTTTTCATTGCAACTTCACCTATTCACATGAAATATAAACTTA780                AAATCAGTCCCGAAGAAGTTTTGAAAAATATTGATAAGTGTGTGAGATACGCACGTGAAC840                GGGTCGAGGTTGTTGAGTTTTCTCCAGAGGATGCAACAAGAACGGAGTTGAATTTTCTTT900                TAGAGGCTGTTCAAACGGCTGTCGATGCTGGAGCAACTTATATTAATATTCCTGACACTG960                TCGGTTATACGACACCAGAAGAATATGGAAAAATTTTTAAATTTTTGATTGATAATACTA1020               AGTCTGACCGAGAAATTATTTTTAGTCCACATTGTCATGATGATTTAGGAATGGCTGTAG1080               CTAATTCATTAGCTGCAATTAAAGCTGGGGCTGGGAGAGTTGAAGGAACTGTCAATGGTA1140               TTGGAGAGCGAGCTGGGAATGCTGCTCTTGAAGAAATTGCTGTGGCACTACATATTCGTA1200               AAGATTTTTATCAGGCACAAAGTCCTTTAAAACTTTCAGAAACTGCTGCAACGGCAGAAC1260               TAATTTCACAATTTTCAGGAATTGCTATTCCAAAAAATAAAGCAATTGTTGGTGCTAATG1320               CTTTTGCACACGAATCAGGAATTCATCAAGATGGTGTCCTTAAAAATGCTGAAACTTATG1380               AAATTATTACACCAGAACTTGTCGGAATAAAGCATAATTCGTTGCCTTTAGGTAAACTTT1440               CTGGTCGTCATGCTTTTAGTGAAAAATTGACGGAACTTAATATTGCTTATGACGATGAAA1500               GTCTTGCAATTTTATTTGAAAAATTTAAAAAATTAGCTGACAAGAAAAAAGAAATTACTG1560               ACGCAGATATTCATGCCTTGTTTACAGGAGAAACGGTAAAAAATCTAGCTGGATTTATAC1620               TTGATAATGTTCAAATTGATGGGCACAAGGCATTGGTGCAACTAAAAAATCAAGAAGAGG1680               AAATTTATGTTAGCCAAGGAGAGGGGTCAGGTTCAGTGGATGCAATTTTTAAAGCTATTG1740               ATAAAGTCTTTAATCATCAACTAAAATTAATTTCCTATTCAGTTGATGCTGTAACTGATG1800               GAATTGATGCACAAGCAACGACTTTGGTTTCTGTTGAAAATCTATCTACAGGCACTATAT1860               TTAATGCTAAAGGTGTTGATTATGATGTATTGAAAGGAAGCGCCATTGCTTACATGAACG1920               CTAATGTTTTAGTTCAAAAAGAAAATTTACAAGGAAAGGTTGAACAAATTTCAGCTCATG1980               ATGGAATTTAAGGTGAAAAATATTGTCTAAAAAAATTGTGACACTTGCGGGAGATGGAAT2040               TGGGCCAGAAATTATGTCAGCTGGTTTAAGTGTTTTAAAAGCTGTCAGTAAAAAAATTGA2100               TTTTGAGTATGAATTAGAAGCTAAAGATTTTGGAGGAATTGCAATTGATAAGCATGGTCA2160               TCCTTTACCAGAAGAAACTTTGCAAGCAGTTAAAAATGCTGACGCAATCTTGCTCGCTGC2220               AATTGGTCATCCTAAATACAACAATGCAAAAGTTAGACCAGAACAAGGGCTACTTGCTTT2280               ACGAAAAGAATTAGGACTGTATGCTAATGTTCGTCCATTAAAAATTTATCCGGCTCTAAA2340               AAAACTTTCTCCCATACGAAATGTTGAAAATGTTGATTTCCTAGTGATTCGCGAACTTAC2400               AGGGGGAATCTATTTCGGTCAGCATGAATTGGCAGATGATAAAGCACGAGATGTCAATGA2460               TTATTCTGCTGATGAAATAAGGAGAATTCTTCATTTTGCTTTCAAAAGTGCTCAAAGTCG2520               GCCCAGAAAATTACTGACTTCGGTTGATAAACAAAATGTTCTTGCAACTTCTAAATTATG2580               GCGAAAAATGGCTGATGAAATTGCTGACGAATATCCTGATGTACGATTAGAGCACCAATT2640               GGTCGATTCTTGTGCGATGTTACTGATTACTAATCCGCAACAATTTGATGTGATAGTCAC2700               TGAAAATCTATTTGGTGATATTCTCTCTGATGAAGCAAGTAGTTTGGCCGGTAGCTTAGG2760               AGTGATGCCTTCGAGTTCGCATGGATTTAACGGTTTAGCACTCTATGAGCCAATTCATGG2820               TTCGGCACCAGATATTGCAGGAAAAGGAATTGCGAACCCTGTTTCGATGATTCTATCAAT2880               TGCCATGATGCTAAGAGAATCTTTTGGGCAAGAAGATGGGGCTGCGATGATTGAAAAAGC2940               CGTAACCCAAACTTTTACTGACGGAATTTTGACTAAAGATTTAGGTGGGACTGCAACAAC3000               TAAAGAAATGACAGAAGCAATCCTGAAAAATTGTCAGTAAAATGCGATTGAATAGTGAGC3060               ATTTTAGTTGTAGATAAAAGAACCGTCAGCATAGCTGACAATTCTGTCAGTAAATGCGAT3120               TGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACCGTCAGCATAGCTGACAATTCTGTC3180               AGTAATTGCGATTGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACCGTCAGCATAGCT3240               GACAATTCTGTCAGTAATTGCGATTGAATAGTGAGCATTTTAGTTGTAGATAAAAGAACT3300               ATCAGCGTAACTGACAATTCTGTCAGTAAATATTACTGACAAAAAGTACAAAATTACTGA3360               CAGAATTTGTCAGAATAAATTTTTAAAAAAGGAAATAAAAAAATGTCAGGTAAAACAATA3420               TTTGATAAACTTTGGGATCAGCATGTGATTGCTGGAAATGAGGGAGAACCTCAACTGCTT3480               TATATTGACCTTCATGTTATTCATGAGGTTACGAGTCCGCAAGCATTTCAGGGCTTACGT3540               GAAGCAGGACGTCGTGTTCGGAGAAAAGATTTGACATACGGAACTCTTGACCACAATGTT3600               CCAACACAAAATATTTTTAATATTCAAGATTTGATTTCTAAAAAACAAATTGATACTTTT3660               ACTAAAAATGTCAAAGAATTTGATGTTCCAGCGGAGACTCATGGTGGAAAAGGACAAGGA3720               ATTGTTCACATGGTAGCACCTGAATCTGGCAGAACTCAACCGGGAAAAACAATTGTTTGT3780               GGCGATAGTCATACCGCAACAAATGGAGCATTTGGTGCAATTGCTTTTGGAATTGGTACA3840               AGTGAAGTTGAACATGTTCTTGCAACTCAAACCATTTGGCAAGTTAAACCCAAGCGTATG3900               AAAATTGAATTTCAAGGTCATCCACAAAAAGGAATTTATAGCAAAGACTTTATCCTCGCA3960               TTAATTGCTAAATATGGTGTGGATGCAGGTGTAGGTTATGCGGTTGAATATAGTGGGGAT4020               GCTATCAGTGATTTAAGCATGGAAGAACGGATGACAATCTGTAACATGTCAATTGAATTT4080               GGGGCAAAAATTGGCCTGATGAATCCTGATGAAAAAACTTATGACTATGTCAAAGGGCGT4140               GAACATGCACCTAAAAACTTTGATGAAGCTGTCAGTAAATGGGAAAAACTTGTCAGTGAT4200               TCTGATGCACAATACGATAAGATTTTAAGTCTTGATGTCAGCCAGTTGAAACCAATGGTG4260               ACATGGGGAACAAATCCCGGAATGGGCCTAGAATTTGGCGAAAAGTTTCCGGAAATTAAC4320               AATGATTTGAATTATGAACGTGCTTATCAGTACATGGATTTAAAGCCAGGCCAAACCGCT4380               TCTGACATAGATTTAGGCTATATTTTCATTGGTTCTTGTACGAATGCTAGACTTGGTGAT4440               TTAGAAGAAGCTGCAAAAATTATTGGAGACAGACATATTGCTGATGGACTGACAGGAATT4500               GTCGTCCCTGGAAGCAGACCTGTGAAAGAAGCGGCTGAAGCACAAGGGCTTGATAAAATT4560               TTTAAAGAAGCTGGTTTTGAATGGCGGGAACCGGGTTGCTCAGCCTGTCTTGGAATGAAT4620               CCTGACCAAATTCCAGAATACGTTCATTGTGCTTCAACCTCTAATCGAAATTTTGAAGGT4680               CGTCAAGGACATAATGCAAGAACGCACCTGTGCTCTCCAGCTATGGCTGCTGCCGCCGCA4740               ATCGCTGGTAAATTTGTAGATGTTAGAATGCTCGTAACAGATTAGTCTGTAGAAAGAAAA4800               AAAGATGGAAAAATTCACGATTTACAAAGGGACAAGTGTTCCAGTCATGAACGATAATAT4860               TGACACAGACCAAATTATTCCTAAACAATTTTTGAAAGCAATCGATAAAAAGGGCTTTGG4920               GAAAAATTTATTTTATGAATGGCGTTATCTTAAAGATTACGATGAGAATCCTGATTTTAT4980               TTTGAATGCTCCAAAATACAAAAAAGCTTCTCTGTTAATTTCAGGAGATAATTTTGGTTC5040               GGGTTCTTCAAGAGAACATGCGGCATGGGCCTTATCAGATTACGGCTTTCGGGCAATTAT5100               TGCTGGCTCTTACTCAGATATTTTTTATAATAATGCTTTAAAAAATGGCTTGTTACCAAT5160               TAAACAACCAAGAGAAGTTCTAAATCAACTGACAAAACTGTCAAGTCAAGAAGAAATTAC5220               AATTGATTTACCCCATCAGCTAATCATCACAAGCCTTGGTGACTTTCATTTTGAGATTGA5280               CCCCATTTGGAAAGACAAATTAATTAATGGCTTAGATGATATTGGAATAACTTTGCAATA5340               TGAAGAAGCAATCTCAGCTTACGAACAAAAAAATCAATAAGAGCGAGCCTAAAATGACAA5400               TTATTAATTTAAAGAATGTAAATCTTACTCGAAATAAAAAAGAAATTCTTAAAGATATTA5460               CTTGGAAAGTAAATCCCGGCGAAAATTGGGTTATTCTGGGCCTCAACGGCTCTGGAAAAT5520               CAAGTCTTTTGAAATTGATTTTAGCAGAAGAATGGAAAACTTCTGGTGAAATCACTGTTT5580               TAAATACTCAATTTAGAAATGGAGAAATTCCTAAGTTGAGAAAAAGAATCAGCGTAGTTG5640               GCTCATTTATTGCTGAAAGATTTCAACCAAATATTAAGGCTGAAAACCTTGTTTATACTG5700               GGAAATTTAATTCGAGCATGCTCTATAAACCCTACACAGATCAGGAACTTGATGAGGCCC5760               GTCAGCTTTTAAGACAAATGGGCGCAAAATCACTTATTGGCCGAAATTATGCCAGCCTTT5820               CTCAAGGGGAAAAGCAAGTTCTTCTTATTGCTAGGAGCTTAATTTTAAAGCCTGAGCTTT5880               TAATTTTGGACGAAGCAACGAACGGTTTAGATTTATTTGCTAAAGAAAAATTATTAAAGC5940               AACTGCAGCAGATTAATCAATTAAAAACCGCACCAACACTAATTTATATTTCTCATCATC6000               CCGATGAAATCACTGATATTTTTACTCACCTTTTACTTTTAAGAGAAGGAAAAGTGATTC6060               AATCAGGGAAAAAAGAAAACTTATTAAATGAAAAGATACTTACTGATTTTTATCAAGAAA6120               AAGTAGAAGTTCACCGTTTTGAGCAGAAATATTTTGTAATTCCTGCTAACTGAGAAAGGA6180               AAGCAAAAGTATTTTATATACTATATAGAATATTCTGACAGATTATTGTATTTTCATTTT6240               TTTAGTGATAAAATAGCTCTATGTAAATTTACGGGGAGGTCAAAAAGATAACATATGGAA6300               TTCAAATATAACGGAAAAGTTGAATCAGTGGAACTCAATAAATATTCTAAGACATTGACT6360               CCAAGATCAACACAACCAGCGACTCAAGCGATGTACTACGGCATTGGTTTTAAAGATGAG6420               GATTTCAAAAAAGCTCAGGTCGGAATCGTCAGCATGGATTGGGACGGAAATCCATGTAAT6480               ATGCACTTGGGAACACTTGGGAGTAAAATCAAAAGTTCTGTCAACCAAACTGACGGATTG6540               ATTGGACTTCAATTTCATACTATTGGAGTTTCTGATGGAATTGCTAACGGAAAGCTTGGC6600               ATGAGATATTCTTTGGTCAGTCGTGAAGTTATTGCTGACAGCATCGAAACCAACGCTGGC6660               GCAGAATATTATGATGCCATCGTTGCCATTCCCGGTTGTGATAAAAATATGCCCGGGTCA6720               ATTATCGGAATGGCTCGCTTAAATCGTCCGTCAATTATGGTCTATGGTGGAACGATTGAA6780               CATGGCGAATATAAAGGTGAAAAATTAAATATTGTTTCGGCCTTTGAAGCTCTGGGGCAA6840               AAAATCACTGGAAATATTTCTGATGAAGATTATCATGGCGTTATTTGCAATGCCATTCCA6900               GGACAAGGTGCTTGCGGAGGAATGTACACTGCCAATACCCTGGCTGCTGCTATTGAAACT6960               TTGGGAATGAGTTTACCTTATTCCTCTTCCAATCCAGCAGTCAGTCAAGAAAAACAAGAA7020               GAGTGTGATGACATTGGTTTAGCCATCAAAAATTTATTAGAAAAAGATATTAAACCAAGT7080               GATATCATGACCAAAGAAGCTTTTGAAAATGCCATAACAATTGTCATGGTCCTTGGAGGC7140               TCAACCAATGCTGTGCTTCATATCATTGCAATGGCAAATGCCATTGGTGTAGAAATTACG7200               CAAGATGATTTCCAACGTATTTCAGATATTATCCCTGTTCTTGGCGATTTCAAACCGAGC7260               GGAAAATATATGATGGAAGATCTGCACAAAATTGGTGGCCTTCCTGCTGTTTTGAAATAC7320               CTACTTAAAGAAGGAAAACTTCACGGTGATTGTTTGACCGTCACAGGTAAAACTTTGGCT7380               GAAAATGTTGAAACAGCATTAGATTTGGACTTTGACAGTCAAGATATTATGCGACCACTA7440               AAAAATCCAATTAAAGCTACTGGACATTTACAAATTTTGTACGGTAATCTTGCCCAAGGG7500               GGTTCTGTTGCAAAAATTTCTGGTAAAGAAGGCGAATTTTTCAAAGGAACAGCTCGTGTT7560               TTTGACGGAGAACAACACTTTATCGATGGCATTGAGTCTGGCCGATTGCATGCCGGTGAT7620               GTTGCGGTCATTAGAAATATTGGCCCAGTCGGAGGTCCGGGAATGCCAGAGATGTTAAAA7680               CCAACCTCAGCATTAATTGGAGCAGGACTTGGAAAATCTTGTGCCCTAATTACTGACGGA7740               AGATTTTCTGGTGGCACACACGGCTTTGTTGTGGGTCATATCGTCCCTGAAGCAGTTGAA7800               GGTGGGTTGATTGGTTTAGTTGAAGATGATGATATTATCGAAATTGATGCGGTGAATAAT7860               AGTATTAGTTTAAAAGTTTCTAATGAAGAAATTGCTAAACGACGTGCCAATTATCAAAAA7920               CCAACCCCTAAAGCAACGCGTGGTGTTCTTGCAAAATTTGCCAAACTTACGCGCCCCGCT7980               AGTGAAGGTTGCGTTACAGATTTATAGAAAGGTTTGAATGAAAAAAATAAAGTTAGAAAA8040               ACCTACTTCCGGTTCCCAACTTGTTCTCCAAACCTTAAAAGAACTTGGAGTAGAAATTAT8100               TTTTGGTTATCCTGGTGGGGCCATGCTCCCCTTGTATGATGCGATTCATAATTTTGAAGG8160               AATTCAACATATTTTAGCCCGTCATGAGCAAGGAGCAACGCATGAAGCCGAAGGTTACGC8220               TAAATCGTCTGGTAAAGTTGGTGTCGTCGTTGTTACGTCAGGACCAGGAGCGACTAATGC8280               AGTAACCGGAATTGCTGACGCTTATCTTGATTCAGTCCCATTGTTAGTTTTCACAGGTCA8340               AGTTGGCCGTCAGTCAATTGGTAAAGATGCTTTTCAAGAAGCAGATACTGTTGGAATTAC8400               AGCCCCAATTACAAAATATAATTATCAAATTAGGGAAACCGCAGATATTCCAAGAATTGT8460               TACAGAAGCCTATTATTTGGCAAGGACAGGACGTCCTGGACCAGTAGAAATTGATTTACC8520               AAAAGATGTTTCCACCCTTGAAGTCACTGAAATTAATGACCCAAGCTTGAATCTTCCTCA8580               TTATCACGAAAGTGAAAAAGCGACTGATGAACAATTGCAAGAATTACTGACAGAACTTTC8640               TGTCAGTAAAAAACCAGTCATTATTGCTGGCGGAGGAATTAATTATTCTGGCTCAGTTGA8700               TATTTTCAGAGCATTTGTCGAAAAATATCAAATTCCAGTTGTTTCTACATTGCTTGGCTT8760               AGGAACATTACCAATCAGCCACGAATTGCAACTAGGAATGGCAGGAATGCACGGTTCATA8820               CGCTGCAAATATGGCTTTAGTTGAAGCTGACTATATTATTAATTTGGGATCACGTTTTGA8880               CGATAGAGTTGTATCCAATCCTGCAAAATTTGCTAAAAATGCTGTCGTTGCTCATATTGA8940               TATTGACGCTGCTGAACTTGGCAAAATTGTAAAAACCGATATTCCAATCCTTTCTGATTT9000               GAAAGCGGCTTTAAGCAGACTTTTGCAATTAAATAAGGTCAGGACTGACTTTAATGATTG9060               GATTAAAACTGTCATTGAAAATAAAGAGAAAGCACCATTTACTTATGAGCCCCAAAACCA9120               TGATATCCGTCCACAGGAAACAATTAAATTAATTGGAGAATACACTCAAGGAGATGCAAT9180               CATTGTAACTGACGTTGGGCAACATCAAATGTGGGTGGCGCAATATTATCCTTATAAAAA9240               TGCAAGGCAACTTATTACTTCTGGGGGAATGGGAACGATGGGCTTTGGCATTCCTGCAGC9300               AATCGGTGCAAAGCTGGCACAGCCAAATAAAAATGTCATTGTTTTTGTTGGCGATGGTGG9360               CTTTCAAATGACTAATCAAGAATTAGCATTACTTAATGGCTACGGTATTGCAATCAAAGT9420               TGTGCTGATTAATAATCATTCATTGGGAATGGTACGTCAATGGCAAGAATCATTCTATGA9480               AGAGCGACGTTCACAATCGGTTTTTGATGTTGAACCCAATTTTCAATTGTTAGCCGAAGC9540               TTATGGCATCAAACATGTTAAGTTAGATAATCCAAAAACTTTGGCTGATGATTTAAAAAT9600               TATTACAGAAGATGAGCCAATGCTTATTGAAGTTCTAATTTCAAAATCTGAGCATGTTTT9660               ACCAATGATACCAGCTGGATTACACAATGACGAAATGATTGGACTTCATTTTACTGATAA9720               GAATGAGGAGATAGATAATGCGTAGAATGATTATCGCAAAACTTCATAACGTGACAGGAA9780               TTATGAATCGATTTACCGCCGTTCTCAATCGAAGGCAAGTGAACATTCTCTCAATTACCG9840               CTGGAGTTACAGAAAGTCAAGACTTAACTCATACCACTTTTGTTATTGAAGTTGATCATC9900               TTGATGAAGTAGAACAAATCATTAAACAATTAAATCGCTTAATAGATGTAATTGAAGTAG9960               CTGATATTACTGATTTTCCTCATGTAGAACGTGAAGTCGTCTTGATTAAAGTATCAGCTC10020              CACCGACCATTAGGGCAGAAATTTTTACAATGATTGAACCTTTTAGAGTAAATGTAGTTG10080              ATGTCAATCTGGAAAATGTCACCATTCAATTAACGGGTGATTCAGCAAAAATCGAAGCAC10140              TTATTGAGGTTGTTAGTCCTTATGGCATTCTAAATATGGCTCGGACAGGTAGTGCAGGTT10200              TTGAGCGTGGCTAAATTTAAATAAGTTAACAAATAAATAGAAAAATAGAGGAAACAAAAA10260              TGGCAGTTACAATGTATTATGAAGATGATGTAGAAGTATCAGCACTTGCTGGAAAGCAAA10320              TTGCAGTAATCGGTTATGGTTCACAAGGACATGCTCACGCACAGAATTTGCGTGATTCTG10380              GTCACAACGTTATCATTGGTGTGCGCCACGGAAAATCTTTTGATAAAGCAAAAGAAGATG10440              GCTTTGAAACATTTGAAGTAGGAGAAGCAGTAGCTAAAGCTGATGTTATTATGGTTTTGG10500              CACCAGATGAACTTCAACAATCCATTTATGAAGAGGACATCAAACCAAACTTGAAAGCAG10560              GTTCAGCACTTGGTTTTGCTCACGGATTTAATATCCATTTTGGCTATATTAAAGTACCAG10620              AAGACGTTGACGTCTTTATGGTTGCGCCTAAGGCTCCAGGTCACCTTGTCCGTCGGACTT10680              ATACTGAAGGTTTTGGTACACCAGCTTTGTTTGTTTCACACCAAAATGCAAGTGGTCATG10740              CGCGTGAAATCGCAATGGATTGGGCCAAAGGAATTGGTTGTGCTCGAGTGGGAATTATTG10800              AAACAACTTTTAAAGAAGAAACAGAAGAAGATTTGTTTGGAGAACAAGCTGTTCTATGTG10860              GAGGTTTGACAGCACTTGTTGAAGCCGGTTTTGAAACACTGACAGAAGCTGGATACGCTG10920              GCGAATTGGCTTACTTTGAAGTTTTGCACGAAATGAAATTGATTGTTGACCTCATGTATG10980              AAGGTGGTTTTACTAAAATGCGTCAATCCATCTCAAATACTGCTGAGTTTGGCGATTATG11040              TGACTGGTCCACGGATTATTACTGACGAAGTTAAAAAGAATATGAAGCTTGTTTTGGCTG11100              ATATTCAATCTGGAAAATTTGCTCAAGATTTCGTTGATGACTTCAAAGCGGGGCGTCCAA11160              AATTAATAGCCTATCGCGAAGCTGCAAAAAATCTTGAAATTGAAAAAATTGGGGCAGAGC11220              ACGTCAAGCAATGCCATTCACACAATCTGGTGATGACGATGCCTTTAAAATCTATCAGTA11280              ATTTCTCTTATTGATTGAACAAAAACATAAAAGCATTTTATGGAGGAATGACATAAATGA11340              TAAGTGCCAAAGAGGTTGAAGATGCCTATGATTTGTTAAAAGCAGTTGTCACTAAAACAC11400              CTTTACAATTAGACCCTTACCTTTCCAATAAATATCAAGCAAATATTTACTTAAAAGAAG11460              TTGTCACTAAAACACCTTTACAATTAGACCCTTACCTTTCCAATAAATATCAAGCAAATA11520              TTTACTTAAAAGAAGAAAACTTACAGAAAGTTCGTTCTTTTAAATTACGAGGAGCTTATT11580              ATTCTATCAGTAAATTATCTGATGAGCAACGCTCTAAAGGAGTGGTTTGTGCCTCAGCAG11640              GAAATCATGCACAAGGGGTTGCTTTTGCTGCAAATCAATTAAATATTTCTGCGACAATTT11700              TTATGCCCGTTACCACACCTAACCAAAAAATTTCACAAGTTAAATTTTTTGGCGAAAGTC11760              ACGTAACAATTCGTTTAATTGGTGATACTTTTGATGAATCAGCCAGAGCAGCAAAAGCTT11820              TTTCTCAAGATAATGACAAACCATTTATAGACCCTTTTGATGATGAAAATGTAATTGCTG11880              GTCAAGGGACAGTGGCTTTAGAAATTTTTGCGCAAGCTAAAAAACAAGGAATAAGTTTAG11940              ATAAGATTTTTGTACAGATTGGTGGAGGTGGTTTAATTGCAGGAATTACTGCCTACAGTA12000              AGGAGCGCTATCCCCAAACTGAAATTATCGGAGTTGAAGCAAAAGGGGCAACAAGTATGA12060              AAGCTGCCTACTCTGCTGGTCAGCCCGTCACCTTGGAACACATTGATAAATTTGCTGACG12120              GAATTGCGGTTGCGACTGTCGGTCAGAAAACTTACCAACTTATTAATGACAAAGTGAAAC12180              AATTGCTTGCGGTTGATGAAGGTTTAATTTCTCAAACCATACTCGAATTGTATTCAAAAT12240              TAGGAATTGTCGCCGAGCCAGCAGGTGCAACATCTGTTGCCGCACTTGAACTTATTAAAG12300              ATGAAATCAAGGGTAAAAATATCGTCTGTATCATCAGCGGCGGAAATAATGATATTAGTC12360              GAATGCAAGAAATTGAAGAAAGAGCTTTGGTTTATGAAGGTCTAAAACATTATTTTGTCA12420              TTAACTTTCCTCAAAGACCAGGATCCTTACGAACTTTTGTCAGTGATATTTTAGGGCCAA12480              ATGATGATATCACCCGATTTGAGTACATCAAAAGGGCTGATAAAGGTAAAGGACCTTGTC12540              TTGTTGGGATTTTACTTTCAGATGCTAGTGATTATGATTCATTGATTAATCGGATTGAAA12600              GATTTGATAATCGTTATGTTAACTTACGTGGAAATGATAGTTTATACGAACTTTTGGTCT12660              AACTAACCAATTGGTTTGAGCCATTTTCTAGTTTCAATTCTCTTTAAATCACTAGAAATT12720              (2) INFORMATION FOR SEQ ID NO: 12:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1728 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ilvB                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                      ATGAAAAAAATAAAGTTAGAAAAACCTACTTCCGGTTCCCAACTTGTTCTCCAAACCTTA60                 AAAGAACTTGGAGTAGAAATTATTTTTGGTTATCCTGGTGGGGCCATGCTCCCCTTGTAT120                GATGCGATTCATAATTTTGAAGGAATTCAACATATTTTAGCCCGTCATGAGCAAGGAGCA180                ACGCATGAAGCCGAAGGTTACGCTAAATCGTCTGGTAAAGTTGGTGTCGTCGTTGTTACG240                TCAGGACCAGGAGCGACTAATGCAGTAACCGGAATTGCTGACGCTTATCTTGATTCAGTC300                CCATTGTTAGTTTTCACAGGTCAAGTTGGCCGTCAGTCAATTGGTAAAGATGCTTTTCAA360                GAAGCAGATACTGTTGGAATTACAGCCCCAATTACAAAATATAATTATCAAATTAGGGAA420                ACCGCAGATATTCCAAGAATTGTTACAGAAGCCTATTATTTGGCAAGGACAGGACGTCCT480                GGACCAGTAGAAATTGATTTACCAAAAGATGTTTCCACCCTTGAAGTCACTGAAATTAAT540                GACCCAAGCTTGAATCTTCCTCATTATCACGAAAGTGAAAAAGCGACTGATGAACAATTG600                CAAGAATTACTGACAGAACTTTCTGTCAGTAAAAAACCAGTCATTATTGCTGGCGGAGGA660                ATTAATTATTCTGGCTCAGTTGATATTTTCAGAGCATTTGTCGAAAAATATCAAATTCCA720                GTTGTTTCTACATTGCTTGGCTTAGGAACATTACCAATCAGCCACGAATTGCAACTAGGA780                ATGGCAGGAATGCACGGTTCATACGCTGCAAATATGGCTTTAGTTGAAGCTGACTATATT840                ATTAATTTGGGATCACGTTTTGACGATAGAGTTGTATCCAATCCTGCAAAATTTGCTAAA900                AATGCTGTCGTTGCTCATATTGATATTGACGCTGCTGAACTTGGCAAAATTGTAAAAACC960                GATATTCCAATCCTTTCTGATTTGAAAGCGGCTTTAAGCAGACTTTTGCAATTAAATAAG1020               GTCAGGACTGACTTTAATGATTGGATTAAAACTGTCATTGAAAATAAAGAGAAAGCACCA1080               TTTACTTATGAGCCCCAAAACCATGATATCCGTCCACAGGAAACAATTAAATTAATTGGA1140               GAATACACTCAAGGAGATGCAATCATTGTAACTGACGTTGGGCAACATCAAATGTGGGTG1200               GCGCAATATTATCCTTATAAAAATGCAAGGCAACTTATTACTTCTGGGGGAATGGGAACG1260               ATGGGCTTTGGCATTCCTGCAGCAATCGGTGCAAAGCTGGCACAGCCAAATAAAAATGTC1320               ATTGTTTTTGTTGGCGATGGTGGCTTTCAAATGACTAATCAAGAATTAGCATTACTTAAT1380               GGCTACGGTATTGCAATCAAAGTTGTGCTGATTAATAATCATTCATTGGGAATGGTACGT1440               CAATGGCAAGAATCATTCTATGAAGAGCGACGTTCACAATCGGTTTTTGATGTTGAACCC1500               AATTTTCAATTGTTAGCCGAAGCTTATGGCATCAAACATGTTAAGTTAGATAATCCAAAA1560               ACTTTGGCTGATGATTTAAAAATTATTACAGAAGATGAGCCAATGCTTATTGAAGTTCTA1620               ATTTCAAAATCTGAGCATGTTTTACCAATGATACCAGCTGGATTACACAATGACGAAATG1680               ATTGGACTTCATTTTACTGATAAGAATGAGGAGATAGATAATGCGTAG1728                           (2) INFORMATION FOR SEQ ID NO: 13:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 477 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ilvN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                      ATGCGTAGAATGATTATCGCAAAACTTCATAACGTGACAGGAATTATGAATCGATTTACC60                 GCCGTTCTCAATCGAAGGCAAGTGAACATTCTCTCAATTACCGCTGGAGTTACAGAAAGT120                CAAGACTTAACTCATACCACTTTTGTTATTGAAGTTGATCATCTTGATGAAGTAGAACAA180                ATCATTAAACAATTAAATCGCTTAATAGATGTAATTGAAGTAGCTGATATTACTGATTTT240                CCTCATGTAGAACGTGAAGTCGTCTTGATTAAAGTATCAGCTCCACCGACCATTAGGGCA300                GAAATTTTTACAATGATTGAACCTTTTAGAGTAAATGTAGTTGATGTCAATCTGGAAAAT360                GTCACCATTCAATTAACGGGTGATTCAGCAAAAATCGAAGCACTTATTGAGGTTGTTAGT420                CCTTATGGCATTCTAAATATGGCTCGGACAGGTAGTGCAGGTTTTGAGCGTGGCTAA477                   (2) INFORMATION FOR SEQ ID NO: 14:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2231 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Lactococcus lactis subsp. lactis                                 (C) INDIVIDUAL ISOLATE: ilvBN                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                      ATTTATAGAAAGGTTTGAATGAAAAAAATAAAGTTAGAAAAACCTACTTCCGGTTCCCAA60                 CTTGTTCTCCAAACCTTAAAAGAACTTGGAGTAGAAATTATTTTTGGTTATCCTGGTGGG120                GCCATGCTCCCCTTGTATGATGCGATTCATAATTTTGAAGGAATTCAACATATTTTAGCC180                CGTCATGAGCAAGGAGCAACGCATGAAGCCGAAGGTTACGCTAAATCGTCTGGTAAAGTT240                GGTGTCGTCGTTGTTACGTCAGGACCAGGAGCGACTAATGCAGTAACCGGAATTGCTGAC300                GCTTATCTTGATTCAGTCCCATTGTTAGTTTTCACAGGTCAAGTTGGCCGTCAGTCAATT360                GGTAAAGATGCTTTTCAAGAAGCAGATACTGTTGGAATTACAGCCCCAATTACAAAATAT420                AATTATCAAATTAGGGAAACCGCAGATATTCCAAGAATTGTTACAGAAGCCTATTATTTG480                GCAAGGACAGGACGTCCTGGACCAGTAGAAATTGATTTACCAAAAGATGTTTCCACCCTT540                GAAGTCACTGAAATTAATGACCCAAGCTTGAATCTTCCTCATTATCACGAAAGTGAAAAA600                GCGACTGATGAACAATTGCAAGAATTACTGACAGAACTTTCTGTCAGTAAAAAACCAGTC660                ATTATTGCTGGCGGAGGAATTAATTATTCTGGCTCAGTTGATATTTTCAGAGCATTTGTC720                GAAAAATATCAAATTCCAGTTGTTTCTACATTGCTTGGCTTAGGAACATTACCAATCAGC780                CACGAATTGCAACTAGGAATGGCAGGAATGCACGGTTCATACGCTGCAAATATGGCTTTA840                GTTGAAGCTGACTATATTATTAATTTGGGATCACGTTTTGACGATAGAGTTGTATCCAAT900                CCTGCAAAATTTGCTAAAAATGCTGTCGTTGCTCATATTGATATTGACGCTGCTGAACTT960                GGCAAAATTGTAAAAACCGATATTCCAATCCTTTCTGATTTGAAAGCGGCTTTAAGCAGA1020               CTTTTGCAATTAAATAAGGTCAGGACTGACTTTAATGATTGGATTAAAACTGTCATTGAA1080               AATAAAGAGAAAGCACCATTTACTTATGAGCCCCAAAACCATGATATCCGTCCACAGGAA1140               ACAATTAAATTAATTGGAGAATACACTCAAGGAGATGCAATCATTGTAACTGACGTTGGG1200               CAACATCAAATGTGGGTGGCGCAATATTATCCTTATAAAAATGCAAGGCAACTTATTACT1260               TCTGGGGGAATGGGAACGATGGGCTTTGGCATTCCTGCAGCAATCGGTGCAAAGCTGGCA1320               CAGCCAAATAAAAATGTCATTGTTTTTGTTGGCGATGGTGGCTTTCAAATGACTAATCAA1380               GAATTAGCATTACTTAATGGCTACGGTATTGCAATCAAAGTTGTGCTGATTAATAATCAT1440               TCATTGGGAATGGTACGTCAATGGCAAGAATCATTCTATGAAGAGCGACGTTCACAATCG1500               GTTTTTGATGTTGAACCCAATTTTCAATTGTTAGCCGAAGCTTATGGCATCAAACATGTT1560               AAGTTAGATAATCCAAAAACTTTGGCTGATGATTTAAAAATTATTACAGAAGATGAGCCA1620               ATGCTTATTGAAGTTCTAATTTCAAAATCTGAGCATGTTTTACCAATGATACCAGCTGGA1680               TTACACAATGACGAAATGATTGGACTTCATTTTACTGATAAGAATGAGGAGATAGATAAT1740               GCGTAGAATGATTATCGCAAAACTTCATAACGTGACAGGAATTATGAATCGATTTACCGC1800               CGTTCTCAATCGAAGGCAAGTGAACATTCTCTCAATTACCGCTGGAGTTACAGAAAGTCA1860               AGACTTAACTCATACCACTTTTGTTATTGAAGTTGATCATCTTGATGAAGTAGAACAAAT1920               CATTAAACAATTAAATCGCTTAATAGATGTAATTGAAGTAGCTGATATTACTGATTTTCC1980               TCATGTAGAACGTGAAGTCGTCTTGATTAAAGTATCAGCTCCACCGACCATTAGGGCAGA2040               AATTTTTACAATGATTGAACCTTTTAGAGTAAATGTAGTTGATGTCAATCTGGAAAATGT2100               CACCATTCAATTAACGGGTGATTCAGCAAAAATCGAAGCACTTATTGAGGTTGTTAGTCC2160               TTATGGCATTCTAAATATGGCTCGGACAGGTAGTGCAGGTTTTGAGCGTGGCTAAATTTA2220               AATAAGTTAAC2231                                                                (2) INFORMATION FOR SEQ ID NO: 15:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                      CTAGTGAAGGTTGCGTTACA20                                                         (2) INFORMATION FOR SEQ ID NO: 16:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: NO                                                         (iii) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                      TGCCATTTTTGTTTCCTCTA20     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We claim:
 1. An isolated nucleic acid molecule encoding a subunit of an α-acetolactate synthase, said nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO:
 12. 2. An isolated nucleic acid molecule encoding a subunit of an α-acetolactate synthase, said nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO:
 13. 3. An isolated nucleic acid molecule encoding a subunit of an α-acetolactate synthase, said nucleic acid molecule encoding an amino acid sequence as shown in SEQ ID NO:
 7. 4. An isolated nucleic acid molecule encoding a subunit of an α-acetolactate synthase, said nucleic acid molecule encoding an amino acid sequence as shown in SEQ ID NO:
 8. 5. An isolated subunit of an α-acetolactate synthase having an amino acid sequence as shown in SEQ ID NO:
 7. 6. An isolated subunit of an α-acetolactate synthase having an amino acid sequence as shown in SEQ ID NO:
 8. 7. An isolated α-acetolactate synthase comprising a first subunit having an amino acid sequence as shown in SEQ ID NO: 7 and a second subunit having an amino acid sequence as shown in SEQ ID NO:
 8. 8. The isolated nucleic acid molecule of claim 3 further comprising one or more regulatory elements selected from the group consisting of a promoter and a transcription termination signal.
 9. An expression vector comprising the nucleic acid molecule of claim
 3. 10. A host cell comprising the expression vector of claim
 9. 11. A host cell comprising the nucleic acid molecule of claim
 3. 12. The host cell of claim 11 wherein a gene coding for lactate dehydrogenase and/or a gene coding for alpha-acetolactate decarboxylase of said host cell are inactivated.
 13. The isolated nucleic acid molecule of claim 4 further comprising one or more regulatory elements selected from the group consisting of a promoter and transcription termination signal.
 14. An expression vector comprising the nucleic acid molecule of claim
 4. 15. A host cell comprising the expression vector of claim
 14. 16. A host cell comprising the nucleic acid molecule of claim
 4. 17. The host cell of claim 16 wherein a gene coding for lactate dehydrogenase and/or a gene coding for alpha-acetolactate decarboxylase of said host cell are inactivated. 